COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF PCSK9 EXPRESSION

Info

Publication number: 20260250646
Type: Application
Filed: May 1, 2023
Publication Date: Aug 27, 2026
Applicants: Chroma Medicine, Inc. (Boston, MA), NChroma Bio,Inc. (Boston, MA)
Inventors: Noorussahar Abubucker (Watertown, MA), Ari Friedland (Cambridge, MA), Morgan Maeder (Waban, MA), Vic Myer (Arlington, MA), Frederic Tremblay (Cambridge, MA), Mary Shirley (Allston, MA)
Application Number: 18/861,783

Abstract

This application relates to compositions and methods comprising epigenetic editors for epigenetic modification of PCSK9, as well as nucleic acids and vectors encoding the same. Also disclosed are cells epigenetically modified by the epigenetic editors.

Description

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/337,164, filed May 1, 2022, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF PCSK9 EXPRESSION,” U.S. Provisional Application No. 63/337,167, filed May 1, 2022, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF PCSK9 EXPRESSION,” and U.S. Provisional Application No. 63/355,083, filed Jun. 23, 2022, entitled “COMPOSITIONS AND METHODS FOR EPIGENETIC REGULATION OF PCSK9 EXPRESSION,” the entire disclosure of each of which is hereby incorporated by reference in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (C169870034W000-SEQ-AXW.xml; Size: 1,831,551 bytes; and Date of Creation: May 1, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

Genome editing has been considered a promising therapeutic approach for the treatment of genetic disease for over a decade. However, manipulation on the DNA level using traditional genetic editors remains risky given the potential for undesired double-strand DNA breaks, heterogenous repair (including large and small insertions and deletions at the intended site), and toxicity. In contrast, targeted epigenetic modification offers the potential to alter gene expression without leading to double-strand break-induced genotoxicity.

One promising candidate for epigenetic silencing is the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene. PCSK9 is a key target in the treatment of heart disease, the leading cause of mortality worldwide ((Berberich et al., Nature Rev Cardiol. (2019) 16(1):9-20). The human PCSK9 gene, located on chromosome 1, has approximately 94% and 80% homology with its cynomolgus and mouse counterparts, respectively. The gene has CpG islands in the promoter region and is distal from other genes and cis-regulatory features. The PCSK9 protein is produced predominantly by the liver.

In humans, PCSK9 plays a key role in regulating the circulating level of low-density lipoprotein (LDL) particles as a result of its binding to the LDL receptor (LDLR). LDLR reduces the circulating concentration of LDL particles by mediating their endocytosis and degradation in the cell. In the absence of PCSK9, or if the interaction of PCSK9 with LDLR is blocked, the rate of recycling of LDLR to the cell surface is increased and recycled LDLR proteins continue to remove LDL particles from the extracellular fluid (Tombling et al., Atherosclerosis (2021) 330:52-60). By contrast, when the endocytosed LDLR is bound to PCSK9, LDLR is degraded along with its passenger LDL particle. Clinical and genetic studies have established that circulating LDL causes atherosclerotic cardiovascular disease (Ference et al., Eur Heart J (2017) 38:2459-72). In addition, loss-of-function mutations in PCSK9 are associated with low LDL levels (Zhao et al., Am J Hum Genet. (2006) 79(3):514-23). Genetic or pharmacologic reduction of PCSK9 decreases cardiovascular events (Ference et al., N Engl J Med. (2016) 375(22):2144-53; Sabatine et al., N Engl J Med. (2017) 376(18):1713-22). Lowering PCSK9 expression can help to increase the recycling of LDLR, which would lead to lower blood LDL particle concentrations.

In view of the critical role of PCSK9 in the pathogenesis of hypercholesterolemia and cardiovascular disease, there is a need for new and improved therapies that target the expression of PCSK9.

SUMMARY

The present disclosure provides systems and compositions for epigenetic modification (“epigenetic editors” or “epigenetic editing systems” herein), and methods of using the same to generate epigenetic modification at PCSK9, including in host cells and organisms.

In some aspects, the present disclosure provides a system for repressing transcription of a human PCSK9 gene in a human cell, optionally a human hepatocyte, comprising

- a) one or more fusion proteins that collectively comprise
  - a DNA methyltransferase (DNMT) domain and/or a domain that recruits a DNMT, optionally wherein the DNMT domain and/or the recruiter domain comprise a DNMT3A domain and/or a DNMT3L domain, and optionally wherein the recruited DNMT is DNMT3A, and
  - a transcriptional repressor domain, each domain being linked to a DNA-binding domain that binds to a target region in the human PCSK9 gene; or
- b) one or more nucleic acid molecules encoding the one or more fusion proteins.

In some embodiments, the DNA-binding domain binds to a target sequence in SEQ ID NO: 1488 or 1489. In certain embodiments, the DNA-binding domain targets the fusion protein(s) to one or more sequences in the PCSK9 gene selected from SEQ ID NOs: 700-747 and 1036-1261.

In some embodiments, the DNA-binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain. For example, the DNA-binding domain may comprise a dCas9 domain, and the system may further comprise (i) one or more guide RNAs (e.g., comprising any one of SEQ ID NOs: 1262-1487), or (ii) nucleic acid molecules coding for the one or more guide RNAs. In certain embodiments, the dCas domain comprises a dCas9 sequence, such as a sequence with at least 90% identity to SEQ ID NO: 12 or 13.

In some embodiments, the fusion protein comprises a dead CRISPR Cas (dCas) domain and the system comprises one or more PCSK9-binding guide RNAs (gRNAs) provided herein. In some embodiments, the system comprises a single gRNA. In some embodiments, the system comprises 2 gRNAs. In some embodiments, the system comprises 3 gRNAs. In some embodiments, the system comprises 4 gRNAs. In some embodiments, the system comprises 5 or more gRNAs. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 2. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 7. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 8. In some embodiments, the system comprises a sgRNA selected from the gRNAs provided in Table 10. In some embodiments, the system comprises a gRNA selected from the gRNAs provided in Table 12.

In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA009 of Table 10, or a gRNA binding the same target domain sequence as gRNA009. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA003 of Table 10, or a gRNA binding the same target domain sequence as gRNA003. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA093 of Table 10, or a gRNA binding the same target domain sequence as gRNA093. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA011 of Table 10, or a gRNA binding the same target domain sequence as gRNA011. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA007 of Table 10, or a gRNA binding the same target domain sequence as gRNA007. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA077 of Table 10, or a gRNA binding the same target domain sequence as gRNA077. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA113 of Table 10, or a gRNA binding the same target domain sequence as gRNA113. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA004 of Table 10, or a gRNA binding the same target domain sequence as gRNA004. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA008 of Table 10, or a gRNA binding the same target domain sequence as gRNA008. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA012 of Table 10, or a gRNA binding the same target domain sequence as gRNA012. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA111 of Table 10, or a gRNA binding the same target domain sequence as gRNA111. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA005 of Table 10, or a gRNA binding the same target domain sequence as gRNA005. In some embodiments, the system comprises a gRNA comprising the gRNA Targeting Sequence of gRNA013 of Table 10, or a gRNA binding the same target domain sequence as gRNA013.

In some embodiments, the system comprises Fusion Protein 9, variant 1, (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 9 variant 2 (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 10 (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 11 (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 12 (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 13 (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 14 (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 15 (Example 12) and gRNA g041. In some embodiments, the system comprises Fusion Protein 9 variant 1 (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein 9 variant 2 (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein 11 (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein 12 (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein 13 (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein 14 (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein 15 (Example 12) and gRNA g049. In some embodiments, the system comprises Fusion Protein 9 variant 1 (Example 12) and gRNAs g041 and g049. In some embodiments, the system comprises Fusion Protein 9 variant 2 (Example 12) and gRNAs g041 and g049. In some embodiments, the system comprises Fusion Protein 10 (Example 12) and gRNAs g041 and g049. In some embodiments, the system comprises Fusion Protein 11 (Example 12) and gRNAs g041 and g049. In some embodiments, the system comprises Fusion Protein 12 (Example 12) and gRNAs g041 and g049. In some embodiments, the system comprises Fusion Protein 13 (Example 12) and gRNAs g041 and g049. In some embodiments, the system comprises Fusion Protein 14 (Example 12) and gRNAs g041 and g049. In some embodiments, the system comprises Fusion Protein 15 (Example 12) and gRNAs g041 and g049.

In some embodiments, the DNA-binding domain comprises a ZFP domain that targets a nucleotide sequence selected from SEQ ID NOs: 700-747. In certain embodiments, the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of ZF001 through ZF048 as shown in Table 1.

In some embodiments, the DNMT3A domain comprises a sequence with at least 90% identity to SEQ ID NO: 574 or 575.

The DNMT3L domain may comprise, e.g., a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 578-581. In some embodiments, the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 582-603. In some embodiments, the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 601-603.

In some embodiments, the transcriptional repressor domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 33-570. In certain embodiments, the transcriptional repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. The KRAB domain may comprise, e.g., a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 89, 116, 245, and 255. In some embodiments, the transcriptional repressor domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 571 or 572. In certain embodiments, the transcriptional repressor domain is derived from KAP1, MECP2, HP1a/CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.

In some embodiments, the system comprises

- a) a fusion protein comprising the DNMT3A domain, the DNMT3L domain, the transcriptional repressor domain, and the DNA-binding domain,
  - optionally wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and
  - optionally wherein the DNA-binding domain is a dead CRISPR Cas domain or a ZFP domain; or
- b) a nucleic acid molecule encoding the fusion protein.

In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, the DNMT3A domain, a first peptide linker, the DNMT3L domain, a second peptide linker, the DNA-binding domain, a third peptide linker, and the transcriptional repressor domain. For example, the fusion protein may comprise, from N-terminus to C-terminus, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, a first nuclear localization signal (NLS), the DNA-binding domain, a second NLS, the third peptide linker, and the transcriptional repressor domain. The fusion protein may comprise, from N-terminus to C-terminus, a first NLS, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and a second NLS. The fusion protein may comprise, from N-terminus to C-terminus, first and second NLSs, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and third and fourth NLSs. In particular embodiments, the transcriptional repressor domain is a KRAB domain, such as a human KOX1, ZFP28, ZN627, or ZIM3 KRAB domain. In particular embodiments, one or both of the second and third peptide linkers are XTEN linkers, which may be selected from XTEN80 (e.g., SEQ ID NO: 643) and XTEN16 (e.g., SEQ ID NO: 638), e.g., wherein the second peptide linker is XTEN80, and the third peptide linker is XTEN16.

In some embodiments, the fusion protein may comprise, from N-terminus to C-terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN16 peptide linker, and a human KOX1 KRAB domain. In certain embodiments, the fusion protein comprises SEQ ID NO: 658 or a sequence at least 90% identical thereto. In certain embodiments, the fusion protein comprises SEQ ID NO: 1495 or a sequence at least 90% identical thereto.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a ZFP domain, a second NLS, an XTEN16 linker, and a human KOX1 KRAB domain. In certain embodiments, the fusion protein comprises SEQ ID NO: 659 or a sequence at least 90% identical thereto, optionally wherein the ZFP comprises, in order, the F1-F6 amino acid sequences of any one of ZF001 through ZF048 as shown in Table 1. In certain embodiments, the fusion protein comprises SEQ ID NO: 1496 or a sequence at least 90% identical thereto, optionally wherein the ZFP comprises, in order, the F1-F6 amino acid sequences of any one of ZF001 through ZF048 as shown in Table 1.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 660 or a sequence at least 90% identical thereto.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 661 or a sequence at least 90% identical thereto.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 662 or a sequence at least 90% identical thereto.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs. In particular embodiments, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 663 or a sequence at least 90% identical thereto.

In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.

In some embodiments, at least one of the NLSs in a fusion protein described herein is an SV40 NLS (e.g., SEQ ID NO: 644).

In some embodiments, the system comprises:

- a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting the DNMT3A domain,
  - a second fusion protein comprising a second DNA-binding domain and comprising or recruiting the DNMT3L domain, and
  - a third fusion protein comprising a third DNA-binding domain and comprising or recruiting the transcriptional repressor domain; or
- b) one or more nucleic acid molecules encoding the fusion proteins.

The present disclosure also provides a human cell comprising a system described herein, or progeny of the cell. In some embodiments, the cell is a hepatocyte.

The present disclosure also provides a pharmaceutical composition comprising a system described herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises lipid nanoparticles (LNPs) comprising the system, and/or the DNA-binding domain is a dCas domain and the LNPs further comprise one or more gRNAs.

The present disclosure also provides a method of treating a patient in need thereof comprising administering a system or pharmaceutical composition described herein to the patient (e.g., intravenously). In some embodiments, the patient has heart disease; has elevated low-density lipoprotein cholesterol (LDL-C) or hypercholesterolemia; is at risk of developing myocardial infarction, stroke, or unstable angina; and/or has primary hyperlipidemia (e.g., heterozygous familial hypercholesterolemia (HeFH), or homozygous familial hypercholesterolemia (HoFH)).

The present disclosure also provides a system or pharmaceutical composition described herein for use in treating a patient in need thereof, e.g., in a method described herein.

The present disclosure also provides use of a system described herein in the manufacture of a medicament for treating a patient in need thereof, e.g., in a method described herein.

The present disclosure also provides articles and kits comprising the systems described herein.

Other features, objectives, and advantages of the disclosed methods and compositions are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and embodiments of the disclosed methods and compositions, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the disclosure will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the predicted binding position of ZF proteins and computationally designed gRNAs on the PCSK9 gene.

FIG. 2 is a scatter plot showing the relative PCSK9 expression (y-axis) at day 7 in cells treated with CRISPR-off (DNMT3A-3L-dCas9-KRAB). The genomic distance from the gRNA target site to the PCSK9 TSS is shown on the x-axis.

FIG. 3 is a diagram showing the overlap of the top 40 gRNAs with the PCSK9 gene.

FIG. 4A is a bar graph showing the level of secreted PCSK9 at day 7 and day 28 following treatment with the indicated gRNA. Dashed line shows silencing achieved by wildtype (WT) Cas9.

FIG. 4B is a scatter plot showing the correlation of PCSK9 mRNA expression and PCSK9 protein secretion in cells following treatment with gRNAs. CRISPRi (dCas9-KRAB) represents a dCas9-KRAB fusion protein.

FIG. 5 is a line graph showing the silencing of PCSK9 following treatment with CRISPRi (dCas9-KRAB), CRISPR-off (DNMT3A-3L-dCas9-KRAB) and the indicated gRNAs.

FIG. 6 is a bar graph showing PCSK9 secretion in cells treated with CRISPRoff and simvastatin, compared to cells treated with the CRISPRoff system alone.

FIG. 7 is a bar graph showing the reduction of PCSK9 secretion in Huh7 hepatoma cells treated with CRISPRoff and the given gRNA.

FIG. 8 is a scatter plot showing the activity and toxicity of 247 PCSK9-targeting ZF proteins. Relative PCSK9 expression is shown on the x-axis and corresponding cell counts relative to the pUC and off-target controls are shown on the y-axis. The diagonal line represents a 1:1 correlation between relative PCSK9 expression and cell count.

FIG. 9 is a scatter plot showing relative PCSK9 expression (y-axis) by cells treated with a ZF-off (DNMT3A-3L-ZF-KRAB) construct and the corresponding targeted genomic distance relative to the PCSK9 transcription start site (TSS) (x-axis).

FIG. 10 is a diagram showing the entire human PCSK9 gene locus flanked with 35.5 kb and 7 kb of upstream and downstream genomic regions (67.5 kb), respectively, that was introduced into and expressed in a transgenic mouse. This transgenic mouse line expresses human PCSK9 under the control of its own (human) endogenous promoter.

FIG. 11A shows schematic illustrations of fusion protein constructs with variant NLS configurations. FIG. 11B shows schematic illustrations of additional fusion protein constructs with variant KRAB domains.

FIGS. 12A-12B are graphs showing the percentage of PCSK9 protein levels measured after treatment with fusion protein constructs with various NLS placements in HeLa cells using 6.25 ng RNA (FIG. 12A) or 2.5 ng RNA (FIG. 12B). Human and murine DNMT3L sequences are indicated as h3L and m3L, respectively.

FIG. 13 is a graph showing that constructs with 2× NLSs are 3× more efficient than CRISPR-off in silencing mPcsk9 in Hepa1-6 cells.

FIG. 14A is a graph showing that constructs with 2× NLSs are more efficient than CRISPR-off in silencing mPcsk9 in Huh7 cells. FIGS. 14B-14C are graphs showing that constructs with 2× NLSs are also more efficient than CRISPR-off in silencing mPcsk9 in Huh7 cells at different doses both at day 5 (FIG. 14B) and day 15 (FIG. 14C).

FIG. 15 is a graph showing that, in Huh7 cells, in a CRISPR-off-like format in which dCas9 is replaced with a zinc finger, 2× NLS offers improvements across multiple ZFs.

FIG. 16 is a set of graphs showing that methylation of the CTLA4 promoter with a bacterial DNMT protein can induce epigenetic silencing of the locus.

FIG. 17 is a set of graphs showing methylation profiles at the VIM3 locus of cells treated with different constructs carrying bacterial DNA methyltransferases fused to dCas9, day 30. Samples treated with M. SssI are methylated by 20%.

FIG. 18 is a set of graphs showing methylation profiles by hybridization capture at the CLTA locus of cells comparing M. SssI to murine DNMT3A/3L in dCas9 fusions, day 29.

FIGS. 19A-19D are a set of graphs showing alternative KRAB domains tested for epi-silencing activity against CRISPR-off when using 0.5 ng effector DNA using CLTA-GFP as a marker (FIG. 19A), 3 ng effector DNA using GFP as a marker (FIG. 19B), and 0.5 ng effector DNA using GFP as a marker (FIG. 19C). FIG. 19D shows results after 30 days using varying nanogram amounts of effector DNA.

DETAILED DESCRIPTION

The present disclosure provides epigenetic editors for regulating expression of the PCSK9 gene. By altering expression of PCSK9, the systems, compositions and methods described herein may be used for treating conditions such as hypercholesterolemia (e.g., heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), familial hypercholesterolemia (HF), or established atherosclerotic cardiovascular disease (ASCVD)), or renal insufficiency (RI). Unless otherwise stated, “PCSK9” refers herein to human PCSK9. A human PCSK9 gene sequence can be found at Ensembl Accession No. ENSG00000169174. The present epigenetic editors have several advantages compared to other genome engineering methods, including reversibility, decreased risk of translocation, and durable, inheritable silencing.

In some embodiments, the region of the human PCSK9 gene targeted for epigenetic regulation is about 2 kb long, and is approximately +/−1 kb of the PCSK9 TSS. In certain embodiments, the region has the nucleotide sequence of SEQ ID NO: 1488. In some embodiments, the targeted PCSK9 region is about 1069 bps long, and is approximately +/−500 bps of the PCSK9 TSS. In certain embodiments, the region targeted has the nucleotide sequence of SEQ ID NO: 1489. The TSS of PCSK9 is at #chr1:55039548 of Genome GRCh38.

In some embodiments, an epigenetic editor as described herein may comprise one or more fusion proteins, wherein each fusion protein comprises a DNA-binding domain linked to one or more effector domains for epigenetic modification. In certain embodiments, where the DNA-binding domain is a polynucleotide guided DNA-binding domain, the epigenetic editor may further comprise one or more guide polynucleotides. DNA-binding domains, effector domains, and guide polynucleotides of an epigenetic editor as described herein may be selected, e.g., from those described below, in any functional combination.

The epigenetic editors described herein may be expressed in a host cell transiently, or may be integrated in a genome of the host cell; such cells and their progeny are also contemplated by the present disclosure. Both transiently expressed and integrated epigenetic editors or components thereof can effect stable epigenetic modifications. For example, after introducing to a host cell an epigenetic editor described herein, the target gene in the host cell may be stably or permanently repressed or silenced. In some embodiments, expression of the target gene is reduced or silenced for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or for the entire lifetime of the cell or the subject carrying the cell, as compared to the level of expression in the absence of the epigenetic editor. The epigenetic modification may be inherited by the progeny of the host cells into which the epigenetic editor was introduced.

The present epigenetic editors may be introduced to a patient in need thereof (e.g., a human patient), e.g., into the patient's hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.

I. DNA-Binding Domains

An epigenetic editor described herein may comprise one or more DNA-binding domains that direct the effector domain(s) of the epigenetic editor to target sequences within or close to the PCSK9 gene locus. A DNA-binding domain as described herein may be, e.g., a polynucleotide guided DNA-binding domain, a zinc finger protein (ZFP) domain, a transcription activator like effector (TALE) domain, a meganuclease DNA-binding domain, and the like. Examples of DNA-binding domains can be found in U.S. Pat. No. 11,162,114, which is incorporated by refence herein in its entirety.

In some embodiments, a DNA-binding domain described herein is encoded by its native coding sequence. In other embodiments, the DNA-binding domain is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells.

A. Polynucleotide Guided DNA-Binding Domains

In some embodiments, a DNA-binding domain herein may be a protein domain directed by a guide nucleic acid sequence (e.g., a guide RNA sequence) to a target site in the PCSK9 gene locus. In certain embodiments, the protein domain may be derived from a CRISPR-associated nuclease, such as a Class I or II CRISPR-associated nuclease. In some embodiments, the protein domain may be derived from a Cas nuclease such as a Type II, Type IIA, Type IIB, Type IIC, Type V, or Type VI Cas nuclease. In certain embodiments, the protein domain may be derived from a Class II Cas nuclease selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas14a, Cas14b, Cas14c, CasX, CasY, CasPhi, C2c4, C2c8, C2c9, C2c10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, and homologues and modified versions thereof “Derived from” is used to mean that the protein domain comprises the full polypeptide sequence of the parent protein, or comprises a variant thereof (e.g., with amino acid residue deletions, insertions, and/or substitutions). The variant retains the desired function of the parent protein (e.g., the ability to form a complex with the guide nucleic acid sequence and the target DNA).

In some embodiments, the CRISPR-associated protein domain may be a Cas9 domain described herein. Cas9 may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence similarity to a wildtype Cas9 polypeptide described herein. In some embodiments, said wildtype polypeptide is Cas9 from Streptococcus pyogenes (NCBI Ref. No. NC_002737.2 (SEQ ID NO: 1)) and/or UniProt Ref No. Q99ZW2 (SEQ ID NO: 2). In some embodiments, said wildtype polypeptide is Cas9 from Staphylococcus aureus (SEQ ID NO: 3). In some embodiments, the CRISPR-associated protein domain is a Cpf1 domain or protein, or a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence similarity to a wildtype Cpf1 polypeptide described herein (e.g., Cpf1 from Franscisella novicida (UniProt Ref. No. U2UMQ6 or SEQ ID NO: 4). In certain embodiments, the CRISPR-associated protein domain may be a modified form of the wildtype protein comprising one or more amino acid residue changes such as a deletion, an insertion, or a substitution; a fusion or chimera; or any combination thereof.

Cas9 sequences and structures of variant Cas9 orthologs have been described for various organisms. Exemplary organisms from which a Cas9 domain herein can be derived include, but are not limited to, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gamma proteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionium, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, and Acaryochloris marina. Cas9 sequences also include those from the organisms and loci disclosed in Chylinski et al., RNA Biol. (2013) 10(5):726-37.

In some embodiments, the Cas9 domain is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 domain is from Staphylococcus aureus (SaCas9).

Other Cas domains are also contemplated for use in the epigenetic editors herein. These include, for example, those from CasX (Cas12E) (e.g., SEQ ID NO: 5), CasY (Cas12d) (e.g., SEQ ID NO: 6), Casp (CasPhi) (e.g., SEQ ID NO: 7), Cas12f1 (Cas14a) (e.g., SEQ ID NO: 8), Cas12f2 (Cas14b) (e.g., SEQ ID NO: 9), Cas12f3 (Cas14c) (e.g., SEQ ID NO: 10), and C2c8 (e.g., SEQ ID NO: 11).

For epigenetic editing, the nuclease-derived protein domain (e.g., a Cas9 or Cpf1 domain) may have reduced or no nuclease activity through mutations such that the protein domain does not cleave DNA or has reduced DNA-cleaving activity while retaining the ability to complex with the guide nucleic acid sequence (e.g., guide RNA) and the target DNA. For example, the nuclease activity may be reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% compared to the wildtype domain. In some embodiments, a CRISPR-associated protein domain described herein is catalytically inactive (“dead”). Examples of such domains include, for example, dCas9 (“dead” Cas9), dCpf1, ddCpf1, dCasPhi, ddCas12a, dLbCpf1, and dFnCpf1. A dCas9 protein domain, for example, may comprise one, two, or more mutations as compared to wildtype Cas9 that abrogate its nuclease activity. The DNA cleavage domain of Cas9 is known to include two subdomains: the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A (in RuvC1) and H840A (in HNH) completely inactivate the nuclease activity of SpCas9. SaCas9, similarly, may be inactivated by the mutations D10A and N580A. In some embodiments, the dCas9 comprises at least one mutation in the HNH subdomain and/or the RuvC1 subdomain that reduces or abrogates nuclease activity. In some embodiments, the dCas9 only comprises a RuvC1 subdomain, or only comprises an HNH subdomain. It is to be understood that any mutation that inactivates the RuvC1 and/or the HNH domain may be included in a dCas9 herein, e.g., insertion, deletion, or single or multiple amino acid substitution in the RuvC1 domain and/or the HNH domain.

In some embodiments, a dCas9 protein herein comprises a mutation at position(s) corresponding to position D10 (e.g., D10A), H840 (e.g., H840A), or both, of a wildtype SpCas9 sequence as numbered in the sequence provided at UniProt Accession No. Q99ZW2 (SEQ ID NO: 2). In particular embodiments, the dCas9 comprises the amino acid sequence of dSpCas9 (D10A and H840A) (SEQ ID NO: 12).

In some embodiments, a dCas9 protein as described herein comprises a mutation at position(s) corresponding to position D10 (e.g., D10A), N580 (e.g., N580A), or both, of a wildtype SaCas9 sequence (e.g., SEQ ID NO: 3). In particular embodiments, the dCas9 comprises the amino acid sequence of dSaCas9 (D10A and N580A) (SEQ ID NO.: 13).

Additional suitable mutations that inactivate Cas9 will be apparent to those of skill in the art based on this disclosure and knowledge in the field and are within the scope of this disclosure. Such mutations may include, but are not limited to, D839A, N863A, and/or K603R in SpCas9. The present disclosure contemplates any mutations that reduce or abrogate the nuclease activity of any Cas9 described herein (e.g., mutations corresponding to any of the Cas9 mutations described herein).

A dCpf1 protein domain may comprise one, two, or more mutations as compared to wildtype Cpf1 that reduce or abrogate its nuclease activity. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9, but does not have an HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. In some embodiments, the dCpf1 comprises one or more mutations corresponding to position D917A, E1006A, or D1255A as numbered in the sequence of the Francisella novicida Cpf1 protein (FnCpf1; SEQ ID NO: 4). In certain embodiments, the dCpf1 protein comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A, or corresponding mutation(s) in any of the Cpf1 amino acid sequences described herein. In some embodiments, the dCpf1 comprises a D917A mutation. In particular embodiments, the dCpf1 comprises the amino acid sequence of dFnCpf1 (SEQ ID NO: 14).

Further nuclease inactive CRISPR-associated protein domains contemplated herein include those from, for example, dNmeCas9 (e.g., SEQ ID NO: 15), dCjCas9 (e.g., SEQ ID NO: 16), dStlCas9 (e.g., SEQ ID NO: 17), dSt3Cas9 (e.g., SEQ ID NO: 18), dLbCpf1 (e.g., SEQ ID NO: 19), dAsCpf1 (e.g., SEQ ID NO: 20), denAsCpf1 (e.g., SEQ ID NO: 21), dHFAsCpf1 (e.g., SEQ ID NO: 22), dRVRAsCpf1 (e.g., SEQ ID NO: 23), dRRAsCpf1 (e.g., SEQ ID NO: 24), dCasX (e.g., SEQ ID NO: 25), and dCasPhi (e.g., SEQ ID NO: 26).

In some embodiments, a Cas9 domain described herein may be a high fidelity Cas9 domain, e.g., comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and the sugar-phosphate backbone of DNA to confer increased target binding specificity. In certain embodiments, the high fidelity Cas9 domain may be nuclease inactive as described herein.

A CRISPR-associated protein domain described herein may recognize a protospacer adjacent motif (PAM) sequence in a target gene. A “PAM” sequence is typically a 2 to 6 bp DNA sequence immediately following the sequence targeted by the CRISPR-associated protein domain. The PAM sequence is required for CRISPR protein binding and cleavage but is not part of the target sequence. The CRISPR-associated protein domain may either recognize a naturally occurring or canonical PAM sequence or may have altered PAM specificity. CRISPR-associated protein domains that bind to non-canonical PAM sequences have been described in the art. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver et al., Nature (2015) 523(7561):481-5 and Kleinstiver et al., Nat Biotechnol. (2015) 33:1293-8. Such Cas9 domains may include, for example, those from “VRER” SpCas9, “EQR” SpCas9, “VQR” SpCas9, “SpG Cas9,” “SpRYCas9,” and “KKH” SaCas9. Nuclease inactive versions of these Cas9 domains are also contemplated, such as nuclease inactive VRER SpCas9 (e.g., SEQ ID NO: 27), nuclease inactive EQR SpCas9 (e.g., SEQ ID NO: 28), nuclease inactive VQR SpCas9 (e.g., SEQ ID NO: 29), nuclease inactive SpG Cas9 (e.g., SEQ ID NO: 30), nuclease inactive SpRY Cas9 (e.g., SEQ ID NO: 31), and nuclease inactive KKH SaCas9 (e.g., SEQ ID NO: 32). Another example is the Cas9 of Francisella novicida engineered to recognize 5′-YG-3′ (where “Y” is a pyrimidine).

Additional suitable CRISPR-associated proteins, orthologs, and variants, including nuclease inactive variants and sequences, will be apparent to those of skill in the art based on this disclosure.

Guide RNAs that can be used in conjunction with the CRISPR-associated protein domains herein are further described in Section II below.

B. Zinc Finger Protein Domains

In some embodiments, the DNA-binding domain of an epigenetic editor described herein comprises a zinc finger protein (ZFP) domain (or “ZF domain” as used herein). ZFPs are proteins having at least one zinc finger, and bind to DNA in a sequence-specific manner. A “zinc finger” (ZF) or “zinc finger motif” (ZF motif) refers to a polypeptide domain comprising a beta-beta-alpha (00a)-protein fold stabilized by a zinc ion. A ZF binds from two to four base pairs of nucleotides, typically three or four base pairs (contiguous or noncontiguous). Each ZF typically comprises approximately 30 amino acids. ZFP domains may contain multiple ZFs that make tandem contacts with their target nucleic acid sequence. A tandem array of ZFs may be engineered to generate artificial ZFPs that bind desired nucleic acid targets. ZFPs may be rationally designed by using databases comprising triplet (or quadruplet) nucleotide sequences and individual ZF amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of ZFs that bind the particular triplet or quadruplet sequence. See, e.g., U.S. Pat. Nos. 6,453,242, 6,534,261, and 8,772,453.

ZFPs are widespread in eukaryotic cells, and may belong to, e.g., C2H2 class, CCHC class, PHD class, or RING class. An exemplary motif characterizing one class of these proteins (C2H2 class) is -Cys-(X)_2-4-Cys-(X)₁₂-His-(X)_3-5-His- (SEQ ID NO: 657), where X is any independently chosen amino acid. In some embodiments, a ZFP domain herein may comprise a ZF array comprising sequential C2H2-ZFs each contacting three or more sequential nucleotides.

A ZFP domain of an epigenetic editor described herein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more ZFs. The ZFP domain may include an array of two-finger or three-finger units, e.g., 3, 4, 5, 6, 7, 8, 9 or 10 or more units, wherein each unit binds a subsite in the target sequence. In some embodiments, a ZFP domain comprising at least three ZFs recognizes a target DNA sequence of 9 or 10 nucleotides. In some embodiments, a ZFP domain comprising at least four ZFs recognizes a target DNA sequence of 12 to 14 nucleotides. In some embodiments, a ZFP domain comprising at least six ZFs recognizes a target DNA sequence of 18 to 21 nucleotides.

In some embodiments, ZFs in a ZFP domain described herein are connected via peptide linkers. The peptide linkers may be, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In some embodiments, a linker comprises or more amino acids. In some embodiments, a linker comprises 7-17 amino acids. The linker may be flexible or rigid.

In some embodiments a zinc finger array may have the sequence:

(SEQ ID NO: 650) SRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXX XXXHXXTH[linker]FQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRIC MRNFSXXXXXXXHXXTH[linker]PFQCRICMRNFSXXXXXXXHXXTHT GEKPFQCRICMRNFSXXXXXXXHXXTHLRGS,

or a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, where “XXXXXXX” represents the amino acids of the ZF recognition helix, which confers DNA-binding specificity upon the zinc finger; each X may be independently chosen. In the above sequence, “XX” in italics may be TR, LR or LK, and “[linker]” represents a linker sequence. In some embodiments, the linker sequence is TGSQKP (SEQ ID NO: 651); this linker may be used when sub-sites targeted by the ZFs are adjacent. In some embodiments, the linker sequence is TGGGGSQKP (SEQ ID NO: 652); this linker may be used when there is a base between the sub-sites targeted by the zinc fingers. The two indicated linkers may be the same or different.

ZFP domains herein may contain arrays of two or more adjacent ZFs that are directly adjacent to one another (e.g., separated by a short (canonical) linker sequence), or are separated by longer, flexible or structured polypeptide sequences. In some embodiments, directly adjacent fingers bind to contiguous nucleic acid sequences, i.e., to adjacent trinucleotides/triplets. In some embodiments, adjacent fingers cross-bind between each other's respective target triplets, which may help to strengthen or enhance the recognition of the target sequence, and leads to the binding of overlapping sequences. In some embodiments, distant ZFs within the ZFP domain may recognize (or bind to) non-contiguous nucleotide sequences.

The amino acid sequences of the ZF DNA-recognition helices of exemplary ZFP domains herein, and their PCSK9 target sequences, are shown below in Table 1, where numbers within the parentheses denote SEQ ID NOs:

TABLE 1 ZF Sequences of Exemplary ZFP Domains Targeting PCSK9 ZF No. DNA Target F1 F2 F3 F4 F5 F6 ZF001 CCGGCAGAACT DHSSLKR QSPHLQR YKHVLVN QMSNLDR QAETLKR RNYDLGI TGGAGTC (748) (749) (750) (751) (752) (753) (700) ZF002 GGTGGGGAGGA QSTTLKR QAHGLTA DAANLRR RRDNLLR RNTHLAR EAHHLSR CTGTGCA (754) (755) (756) (757) (758) (759) (701) ZF003 GGTGGGGAGGA QSTTLKR GAHGLAG DQTNLRR RQDNLQR KGDHLRR EAHHLSR CTGTGCA (760) (761) (762) (763) (764) (765) (702) ZF004 GATGGGGCTCT RTDTLAR RKTALNR RNESLKV VKNTLTR RREHLVR ISHNLAR GGTGGCG (766) (767) (768) (769) (770) (771) (703) ZF005 GATGGGGCTCT RRLTLDR RRDLLHN RNESLKV VGASLKR RREHLVR LEHNLGR GGTGGCG (772) (773) (774) (775) (776) (777) (704) ZF006 GATGGGGCTCT RNHTLQR RREVLEN RHESLIV VGASLKR KKDHLHR LMHNLTR GGTGGCG (778) (779) (780) (781) (782) (783) (705) ZF007 GCAGGAGGATG KRERLER QKGNLVV QKTHLAV QGGHLKR QRPHLTN QAATLQR AAAAGGG (784) (785) (786) (787) (788) (789) (706) ZF008 GGAGCCGCTGC RSQTLAQ QSTTLKR LRDSLKR LGHTLNR DPSVLTR QNSHLRR TGCAACG (790) (791) (792) (793) (794) (795) (707) ZF009 GACTGTGCAGG QRGNLAR VSNTLTR QRPHLTN QAATLQR RRQHLEL DRGNLTR AGCTGAA (796) (797) (798) (799) (800) (801) (708) ZF010 GGGGAGGACTG QSAHLKR QDVSLVR RKQHLQL DRGNLTR RDDNLQR RMEHLPR TGCAGGA (802) (803) (804) (805) (806) (807) (709) ZF011 GGTGGGGAGGA QSTTLKR QPHGLGA LVENLRR RVENLHR RREHLVR LSQGLAR CTGTGCA (808) (809) (810) (811) (812) (813) (710) ZF012 GGTGGGGAGGA QSTTLKR QAHGLTA DGSNLAR RRDNLLR RREHLVR VDHHLRR CTGTGCA (814) (815) (816) (817) (818) (819) (711) ZF013 TGCGGTGGGGA RKQHLQL DRGNLTR RPDNLVR RIDKLGG EAHHLSR QGRSLRA GGACTGT (820) (821) (822) (823) (824) (825) (712) ZF014 GTGGAAGGTGG EAHHLSR QPHGLRA LKEHLTR MGHHLKR QRGNLLR RPDALPR CTGTGGT (826) (827) (828) (829) (830) (831) (713) ZF015 GCGGTGGAAGG RRQHLQY LKEHLTR SKQKLQV QQTNLTR RREVLEN RPDGLAR TGGCTGT (832) (833) (834) (835) (836) (837) (714) ZF016 GCGGTGGAAGG RRQHLTL DNSHLQR LPHHLQR QSNNLTR RNFILQR RKDDLKR TGGCTGT (838) (839) (840) (841) (842) (843) (715) ZF017 GCGGCGGGGGA RQEHLVR EGGNLMR RPDNLVR RIDKLGG RRDDLTR RKDLLHR GGACGGG (844) (845) (846) (847) (848) (849) (716) ZF018 GCGGCGGGGGA RREHLVR DMGNLGR RDDNLQR RMEHLPR RKEDLAR RLDMLAR GGACGGG (850) (851) (852) (853) (854) (855) (717) ZF019 TAGGGATGGGA RREHLVR EHRGLKR RGDNLGR RSDHLSL QQAHLVR RDDNLRT GGCCGGG (856) (857) (858) (859) (860) (861) (718) ZF020 GGCGCCGCCGG DDANLRR RREVLEN LKEHLTR DPSNLRR DSSVLRR ENSKLNR CGTGGAC (862) (863) (864) (865) (866) (867) (719) ZF021 GGCGCCGCCGG DRANLRR RPDALSR LKEHLTR DPSNLRR DSSVLRR ENSKLNR CGTGGAC (868) (869) (870) (871) (872) (873) (720) ZF022 GTCGTTGCAGC LSQTLKR RTDTLAR QMETLKR QGGTLRR HKSSLTR DRTPLQR AGCGGCT (874) (875) (876) (877) (878) (879) (721) ZF023 GATGGGGCTCT RTDTLAR RKTALNR RNESLKV VKNTLTR RREHLVR LTHNLRR GGTGGCG (880) (881) (882) (883) (884) (885) (722) ZF024 TCGGTGGGCAG RLDNLDR RADNLRR SHWKLHT EKGHLNR RREVLEN RADGLQL CGAGGAG (886) (887) (888) (889) (890) (891) (723) ZF025 GGGGAGGACTG QSPHLKR QSTSLQR RKQHLTL DRGNLTR RPHNLLR RREHLVR TGCAGGA (892) (893) (894) (895) (896) (897) (724) ZF026 GGGGAGGACTG QQAHLVR QAETLKR RKQHLTL DRGNLTR RNTNLTR RREHLVR TGCAGGA (898) (899) (900) (901) (902) (903) (725) ZF027 GCGGCGGGGGA RREHLVR DPSNLQR RNTNLTR RREHLVR RTDTLAR RVDDLGR GGACGGG (904) (905) (906) (907) (908) (909) (726) ZF028 GAGGAGTGAGC QSHSLKS ESGHLKR DLSTLRR QNEHLKV RPENLNR RRDNLNR CAGGCAGT (910) (911) (912) (913) (914) (915) (727) ZF029 GGTGTGGGTGC ERRGLDR DRGNLTR LSQTLNR IKHHLGR RNFILQR ERHQLVR TTGACGCC (916) (917) (918) (919) (920) (921) (728) ZF030 TCTGAGCCTGG QREHLVT RIDNLGR RMSNLVR RNESLKV RADNLGR ARNTLKG AGGAGTGA (922) (923) (924) (925) (926) (927) (729) ZF031 GAGGAGTGAGC QSHSLKS EKSHLTR DSPTLRR QKVHLQV RAENLAR RRDNLLR CAGGCAGT (928) (929) (930) (931) (932) (933) (730) ZF032 GAGGGCCAGGG HKSSLTR RPDNLPR QGTHLRN RTHHLIT TPSKLDR RQDNLGR GAGAGGTT (934) (935) (936) (937) (938) (939) (731) ZF033 GCAGGAGGACG ERAKLIR DPSNLRR RQDNLGR DQGNLGR QSAHLKR QDVSLVR AGGACGGC (940) (941) (942) (943) (944) (945) (732) ZF034 GCAGGAGGACG TPSKLDR LAENLRR RQDNLGR DGGNLGR QSPHLKR QSTSLQR AGGACGGC (946) (947) (948) (949) (950) (951) (733) ZF035 GCAGGAGGACG TPSKLDR DSSNLRR RQDNLGR EGGNLMR QQAHLVR QAETLKR AGGACGGC (952) (953) (954) (955) (956) (957) (734) ZF036 TGGGGAGGACT QRPHLTN QAATLQR RKECLVV QNPHLLR QSAHLKR RSDHLSL GTGCAGGA (958) (959) (960) (961) (962) (963) (735) ZF037 GTTGCAGGCGG DPSVLTR EHRGLKR VPSKLKR RDDTLVR QKETLNR VRSSLRR GCGCCGCC (964) (965) (966) (967) (968) (969) (736) ZF038 GTTGCAGGCGG DPSVLTR EHRGLKR KTDHLAR DKAHLVR QKETLNR VRSSLRR GCGCCGCC (970) (971) (972) (973) (974) (975) (737) ZF039 GCTGTTTGGGG RADNLGR KQVTLRN KHSNLTR RREHLVR VKSSLTR VSNSLNR AGGGCGAG (976) (977) (978) (979) (980) (981) (738) ZF040 GCTGTTTGGGG RDDNLQR KNVTLTN QSAHLKR RSDHLSL VKSSLTR VSNSLNR AGGGCGAG (982) (983) (984) (985) (986) (987) (739) ZF041 GCAGAGGCCGG DPSVLKR RTEHLAR QSPHLKR DQTTLRR KHSNLTR QMETLKR AGGGGGTC (988) (989) (990) (991) (992) (993) (740) ZF042 TGGGGAGGGCG QNQNLAR DKSVLAR RDDNLQR KNVTLTN QSAHLKR RSDHLSL AGGCCGAA (994) (995) (996) (997) (998) (999) (741) ZF043 GCGGGTGTAGG KHSNLTR RREHLTI KKDHLHR QTTTLKR EEHHLTR REDVLGR GATGGGAG (1000) (1001) (1002) (1003) (1004) (1005) (742) ZF044 GCGGGTGTAGG KHSNLTR RREHLTI KKDHLHR QTTTLKR EAHHLSR RTDDLGR GATGGGAG (1006) (1007) (1008) (1009) (1010) (1011) (743) ZF045 GGAGCTGGGAG LRQTLAR VAHSLKR DRSVLVR QNSHLRR VKHSLQR QTTHLSR CCGCTGCT (1012) (1013) (1014) (1015) (1016) (1017) (744) ZF046 GCAAGGCGGCG QSAHLKR QMSHLKR RGNHLRR LKEHLTR RNEHLKV QSTTLKR GGGGAGGA (1018) (1019) (1020) (1021) (1022) (1023) (745) ZF047 GCTGTTTGGGG RKPHLDN RPDVLMR RDDNLQR RMEHLPR HQSSLTR VSNSLAR AGGGCGAGG (1024) (1025) (1026) (1027) (1028) (1029) (746) ZF048 GCTGTTTGGGG RNIHLQT RKDTLAR RADNLGR RMEHLPR HQSSLTR VSNSLAR AGGGCGAGG (1030) (1031) (1032) (1033) (1034) (1035) (747)

In some embodiments, the ZFP domain of the present epigenetic editor binds to a target sequence selected from any one of SEQ ID NOs: 700-747. In further embodiments, the ZFP domain comprises, in order, the F1-F6 amino acid sequences of any one of ZF001-ZF048 as shown in Table 1. The F1-F6 amino acid sequences may be placed within the ZF framework sequence of SEQ TD NO: 650, or within any other ZF framework known in the art.

C. TALEs

In some embodiments, the DNA-binding domain of an epigenetic editor described herein comprises a transcription activator-like effector (TALE) domain. The DNA-binding domain of a TALE comprises a highly conserved sequence of about 33-34 amino acids, with a repeat variable di-residue (RVD) at positions 12 and 13 that is central to the recognition of specific nucleotides. TALEs can be engineered to bind practically any desired DNA sequence. Methods for programming TALEs are known in the art. For example, such methods are described in Carroll et al., Genet Soc Amer. (2011) 188(4):773-82; Miller et al., Nat Biotechnol. (2007) 25(7):778-85; Christian et al., Genetics (2008) 186(2):757-61; Li et al., Nucl Acids Res. (2010) 39(1):359-72; and Moscou et al., Science (2009) 326(5959):1501.

D. Other DNA-Binding Domains

Other DNA-binding domains are contemplated for the epigenetic editors described herein. In some embodiments, the DNA-binding domain comprises an argonaute protein domain, e.g., from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease that is guided to its target site by 5′ phosphorylated ssDNA (gDNA), where it produces double-strand breaks. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Thus, using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described, e.g., in Gao et al., Nat Biotechnol. (2016) 34(7):768-73; Swarts et al., Nature (2014) 507(7491):258-61; and Swarts et al., Nucl Acids Res. (2015) 43(10):5120-9.

In some embodiments, the DNA-binding domain comprises an inactivated nuclease, for example, an inactivated meganuclease. Additional non-limiting examples of DNA-binding domains include tetracycline-controlled repressor (tetR) DNA-binding domains, leucine zippers, helix-loop-helix (HLH) domains, helix-turn-helix domains, β-sheet motifs, steroid receptor motifs, bZIP domains homeodomains, and AT-hooks.

II. Guide Polynucleotides

Epigenetic editors described herein that comprise a polynucleotide guided DNA-binding domain may also include a guide polynucleotide that is capable of forming a complex with the DNA-binding domain. The guide polynucleotide may comprise RNA, DNA, or a mixture of both. For example, where the polynucleotide guided DNA-binding domain is a CRISPR-associated protein domain, the guide polynucleotide may be a guide RNA (gRNA). A “guide RNA” or “gRNA” refers to a nucleic acid that is able to hybridize to a target sequence and direct binding of the CRISPR-Cas complex to the target sequence. Methods of using guide polynucleotide sequences with programmable DNA-binding proteins (e.g., CRISPR-associated protein domains) for site-specific DNA targeting (e.g., to modify a genome) are known in the art.

A guide polynucleotide sequence (e.g., a gRNA sequence) may comprises two parts: 1) a nucleotide sequence comprising a “targeting sequence” that is complementary to a target nucleic acid sequence (“target sequence”), e.g., to a nucleic acid sequence comprised in a genomic target site; and 2) a nucleotide sequence that binds a polynucleotide guided DNA-binding domain (e.g., a CRISPR-Cas protein domain). The nucleotide sequence in 1) may comprise a targeting sequence that is 100% complementary to a genomic nucleic acid sequence, e.g., a nucleic acid sequence comprised in a genomic target site, and thus may hybridize to the target nucleic acid sequence. The nucleotide sequence in 1) may be referred to as, e.g., a crispr RNA, or crRNA. The nucleotide sequence in 2) may be referred to as a scaffold sequence of a guide nucleic acid, e.g., a tracrRNA, or an activating region of a guide nucleic acid, and may comprise a stem-loop structure. Parts 1) and 2) as described above may be fused to form one single guide (e.g., a single guide RNA, or sgRNA), or may be on two separate nucleic acid molecules. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a linker. In some embodiments, a guide polynucleotide comprises parts 1) and 2) connected by a non-nucleic acid linker, for example, a peptide linker or a chemical linker.

Part 2 (the scaffold sequence) of a guide polynucleotide as described herein may be, for example, as described in Jinek et al., Science (2012) 337:816-21; U.S. Patent Publication 2016/0208288; or U.S. Patent Publication 2016/0200779. Variants of part 2) are also contemplated by the present disclosure. For example, the tetraloop and stem loop of a gRNA scaffold (tracrRNA) sequence may be modified to include RNA aptamers, which can be bound by specific protein domains. In some embodiments, such modified gRNAs can be used to facilitate the recruitment of repressive or activating domains fused to the protein-interacting RNA aptamers.

A gRNA as provided herein typically comprises a targeting domain and a binding domain. The targeting domain (also termed “targeting sequence”) may comprise a nucleic acid sequence that binds to a target site, e.g., to a genomic nucleic acid molecule within a cell. The target site may be a double-stranded DNA sequence comprising a PAM sequence as well as the target sequence, which is located on the same strand as, and directly adjacent to, the PAM sequence. The targeting domain of the gRNA may comprise an RNA sequence that corresponds to the target sequence, i.e., it resembles the sequence of the target domain, sometimes with one or more mismatches, but typically comprising an RNA sequence instead of a DNA sequence. The targeting domain of the gRNA thus may base pair (in full or partial complementarity) with the sequence of the double-stranded target site that is complementary to the target sequence, and thus with the strand complementary to the strand that comprises the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not include a sequence that resembles the PAM sequence. It will further be understood that the location of the PAM may be 5′ or 3′ of the target sequence, depending on the nuclease employed. For example, the PAM is typically 3′ of the target sequence for Cas9 nucleases, and 5′ of the target sequence for Cas12a nucleases. For an illustration of the location of the PAM and the mechanism of gRNA binding to a target site, see, e.g., FIG. 1 of Vanegas et al., Fungal Biol Biotechnol. (2019) 6:6, which is incorporated by reference herein. For additional illustration and description of the mechanism of gRNA targeting of an RNA-guided nuclease to a target site, see Fu et al., Nat Biotechnol (2014) 32(3):279-84 and Sternberg et al., Nature (2014) 507(7490):62-7, each incorporated herein by reference.

In some embodiments, the targeting domain sequence comprises between 17 and nucleotides and corresponds fully to the target sequence (i.e., without any mismatch nucleotides). In some embodiments, however, the targeting domain sequence may comprise one or more, but typically not more than 4, mismatches, e.g., 1, 2, 3, or 4 mismatches. As the targeting domain is part of gRNA, which is an RNA molecule, it will typically comprise ribonucleotides, while the DNA targeting domain will comprise deoxyribonucleotides.

An exemplary illustration of a Cas9 target site, comprising a 22 nucleotide target domain, and an NGG PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target sequence (and thus base pairs with full complementarity with the DNA strand complementary to the strand comprising the target sequence and PAM) is provided below:

[ target domain (DNA) ][ PAM ] 5′-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-G-G-3′ (DNA) 3′-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-C-C-5′ (DNA) | | | | | | | | | | | | | | | | | | | | | | 5′-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-[ gRNA scaffold]-3′ (RNA) [ targeting domain (RNA) ][ binding domain ]

An exemplary illustration of a Cas12a target site, comprising a 22 nucleotide target domain, and a TTN PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target sequence (and thus base pairs with full complementarity with the DNA strand complementary to the strand comprising the target sequence and PAM) is provided below:

[ PAM ][ target domain (DNA) ] 5′-T-T-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3′ (DNA) 3′-A-A-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-5′ (DNA) | | | | | | | | | | | | | | | | | | | | | | 5′-[gRNA scaffold]-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3′ (RNA) [ binding domain ][ targeting domain (RNA) ]

While not wishing to be bound by theory, at least in some embodiments, it is believed that the length and complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA/Cas9 molecule complex with a target nucleic acid. In some embodiments, the targeting domain of a gRNA provided herein is 5 to 50 nucleotides in length. In some embodiments, the targeting domain is 15 to nucleotides in length. In some embodiments, the targeting domain is 18 to 22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In certain embodiments, the targeting domain fully corresponds, without mismatch, to a target sequence provided herein, or a part thereof. In some embodiments, the targeting domain of a gRNA provided herein comprises 1 mismatch relative to a target sequence provided herein. In some embodiments, the targeting domain comprises 2 mismatches relative to the target sequence. In some embodiments, the target domain comprises 3 mismatches relative to the target sequence.

Methods for designing, selecting, and validating gRNAs are described herein and known in the art. Software tools can be used to optimize the gRNAs corresponding to a target DNA sequence, e.g., to minimize total off-target activity across the genome. For example, DNA sequence searching algorithms can be used to identify a target sequence in crRNAs of a gRNA for use with Cas9. Exemplary gRNA design tools include the ones described in Bae et al., Bioinformatics (2014) 30:1473-5.

Guide polynucleotides (e.g., gRNAs) described herein may be of various lengths. In some embodiments, the length of the spacer or targeting sequence depends on the CRISPR-associated protein component of the epigenetic editor system used. For example, Cas proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the spacer sequence may comprise, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides in length. In some embodiments, the spacer comprises 10-24, 11-20, 11-16, 18-24, 19-21, or 20 nucleotides in length. In some embodiments, a guide polynucleotide (e.g., gRNA) is from 15-100 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length and comprises a spacer sequence of at least 10 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) contiguous nucleotides complementary to the target sequence. In some embodiments, a guide polynucleotide described herein may be truncated, e.g., by 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more nucleotides.

In certain embodiments, the 3′ end of the PCSK9 target sequence is immediately adjacent to a PAM sequence (e.g., a canonical PAM sequence such as NGG for SpCas9). The degree of complementarity between the targeting sequence of the guide polynucleotide (e.g., the spacer sequence of a gRNA) and the target sequence may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In particular embodiments, the targeting and the target sequence may be 100% complementary. In other embodiments, the targeting sequence and the target sequence may contain, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

A guide polynucleotide (e.g., gRNA) may be modified with, for example, chemical alterations and synthetic modifications. A modified gRNA, for instance, can include an alteration or replacement of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage, an alteration of the ribose sugar (e.g., of the 2′ hydroxyl on the ribose sugar), an alteration of the phosphate moiety, modification or replacement of a naturally occurring nucleobase, modification or replacement of the ribose-phosphate backbone, modification of the 3′ end and/or 5′ end of the oligonucleotide, replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker, or any combination thereof.

In some embodiments, one or more ribose groups of the gRNA may be modified. Examples of chemical modifications to the ribose group include, but are not limited to, 2′-O-methyl (2′-OMe), 2′-fluoro (2′-F), 2′-deoxy, 2′-O-(2-methoxyethyl) (2′-MOE), 2′-NH2, 2′ -O-allyl, 2′-O-ethylamine, 2′-O-cyanoethyl, 2′-O-acetalester, or a bicyclic nucleotide such as locked nucleic acid (LNA), 2′-(5-constrained ethyl (S-cEt)), constrained MOE, or 2′-0,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNANC). 2′-O-methyl modification and/or 2′-fluoro modification may increase binding affinity and/or nuclease stability of the gRNA oligonucleotides.

In some embodiments, one or more phosphate groups of the gRNA may be chemically modified. Examples of chemical modifications to a phosphate group include, but are not limited to, a phosphorothioate (PS), phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification. In some embodiments, a guide polynucleotide described herein may comprise one, two, three, or more PS linkages at or near the 5′ end and/or the 3′ end; the PS linkages may be contiguous or noncontiguous.

In some embodiments, the gRNA herein comprises a mixture of ribonucleotides and deoxyribonucleotides and/or one or more PS linkages.

In some embodiments, one or more nucleobases of the gRNA may be chemically modified. Examples of chemically modified nucleobases include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and nucleobases with halogenated aromatic groups. Chemical modifications can be made in the spacer region, the tracr RNA region, the stem loop, or any combination thereof.

Table 2 below lists exemplary gRNA target sequences for epigenetic modification of human PCSK9, as well as the coordinates of the start and end positions of the targeted site on human chromosome 1 (SEQ: SEQ ID NO). The Table also shows the distance from the start coordinate to the TSS coordinate of the PCSK9 gene.

TABLE 2 Exemplary Target Sequences of gRNAs Targeting PCSK9 Chr. 1 gRNA Target Sequence TSS gRNA No. Strand START END (DNA, 5′ to 3′) SEQ Distance gRNA001 + 55039960 55039979 GGTGCTAGCCTTGCGTTCCG 1036 431 gRNA002 + 55039991 55040010 CTGGCCGAAGCACCCGAGCA 1037 462 gRNA003 − 55039529 55039548 TGCGGAAACCTTCTAGGGTG 1038 0 gRNA004 − 55039528 55039547 GCGGAAACCTTCTAGGGTGT 1039 −1 gRNA005 + 55039518 55039537 TCAAGCACCCACACCCTAGA 1040 −11 gRNA006 − 55039514 55039533 GGGTGTGGGTGCTTGACGCC 1041 −15 gRNA007 − 55039513 55039532 GGTGTGGGTGCTTGACGCCT 1042 −16 gRNA008 − 55039512 55039531 GTGTGGGTGCTTGACGCCTG 1043 −17 gRNA009 − 55039610 55039629 ACTGCCTGGCTCACTCCTCC 1044 81 gRNA010 − 55039481 55039500 TCACGCCACCAGAGCCCCAT 1045 −48 gRNA011 + 55039473 55039492 ATCGTCCGATGGGGCTCTGG 1046 −56 gRNA012 + 55039470 55039489 AGGATCGTCCGATGGGGCTC 1047 −59 gRNA013 + 55039464 55039483 TCAGATAGGATCGTCCGATG 1048 −65 gRNA014 + 55039669 55039688 GCGGCTCCCAGCTCCCAGCC 1049 140 gRNA015 − 55039678 55039697 CGGAATCCTGGCTGGGAGCT 1050 149 gRNA016 − 55039679 55039698 GCGGAATCCTGGCTGGGAGC 1051 150 gRNA017 − 55039685 55039704 GGGCGCGCGGAATCCTGGCT 1052 156 gRNA018 − 55039686 55039705 GGGGCGCGCGGAATCCTGGC 1053 157 gRNA019 + 55038610 55038629 TGAGGTCTTTGCAAACAAAG 1054 −919 gRNA020 + 55038654 55038673 CCAGCACCTAGATTCAGAGC 1055 −875 gRNA021 − 55038657 55038676 CCTGCTCTGAATCTAGGTGC 1056 −872 gRNA022 − 55038731 55038750 TCGATACTGGGAAGAAACAA 1057 −798 gRNA023 − 55038743 55038762 CTGGAAGGGCTGTCGATACT 1058 −786 gRNA024 − 55038744 55038763 TCTGGAAGGGCTGTCGATAC 1059 785 gRNA025 − 55038757 55038776 GAGGCTTGCTCTTTCTGGAA 1060 −772 gRNA026 − 55038758 55038777 TGAGGCTTGCTCTTTCTGGA 1061 −771 gRNA027 − 55038762 55038781 GACATGAGGCTTGCTCTTTC 1062 −767 gRNA028 − 55038776 55038795 TGTACATGTGGCATGACATG 1063 −753 gRNA029 + 55038781 55038800 CATGCCACATGTACAATCTG 1064 −748 gRNA030 + 55038786 55038805 CACATGTACAATCTGAGGCC 1065 −743 gRNA031 − 55038788 55038807 CTGGCCTCAGATTGTACATG 1066 −741 gRNA032 − 55038807 55038826 AAAAGGGGAAAGAGAGCTCC 1067 −722 gRNA033 − 55038822 55038841 CCAGGCAGGAGGATGAAAAG 1068 −707 gRNA034 − 55038823 55038842 ACCAGGCAGGAGGATGAAAA 1069 −706 gRNA035 − 55038824 55038843 TACCAGGCAGGAGGATGAAA 1070 −705 gRNA036 + 55038830 55038849 CCTCCTGCCTGGTACACAAT 1071 −699 gRNA037 − 55038833 55038852 CCTATTGTGTACCAGGCAGG 1072 −696 gRNA038 − 55038836 55038855 ACACCTATTGTGTACCAGGC 1073 −693 gRNA039 + 55038840 55038859 GGTACACAATAGGTGTTTAC 1074 −689 gRNA040 − 55038840 55038859 GTAAACACCTATTGTGTACC 1075 −689 gRNA041 + 55038870 55038889 TCCAGTTGATTTCTTGAACA 1076 −659 gRNA042 − 55038874 55038893 ACCATGTTCAAGAAATCAAC 1077 −655 gRNA043 + 55038881 55038900 TCTTGAACATGGTGTGTAAA 1078 −648 gRNA044 + 55038906 55038925 TCTTTGCAAATTGAATCTTC 1079 −623 gRNA045 − 55038970 55038989 AGTTTGCAAAGACGTCATAT 1080 −559 gRNA046 − 55038998 55039017 GATTTATACTACAAAGATTC 1081 −531 gRNA047 + 55039075 55039094 AGTTGGTAAGGTCAGTGTGC 1082 −454 gRNA048 + 55039076 55039095 GTTGGTAAGGTCAGTGTGCA 1083 −453 gRNA049 + 55039093 55039112 GCAGGGTGCATAAAGGGCAG 1084 −436 gRNA050 + 55039097 55039116 GGTGCATAAAGGGCAGAGGC 1085 −432 gRNA051 + 55039100 55039119 GCATAAAGGGCAGAGGCCGG 1086 −429 gRNA052 + 55039137 55039156 TTTAGAAGGCTGCCAGGTTA 1087 −392 gRNA053 − 55039163 55039182 GCCCACCGAATTCTTTCCAC 1088 −366 gRNA054 + 55039167 55039186 AAAGAATTCGGTGGGCAGCG 1089 −362 gRNA055 − 55039196 55039215 CTTCTGAATCAATCCTACTG 1090 −333 gRNA056 + 55039221 55039240 CTGGTCAGCAGGAGACAAGG 1091 −308 gRNA057 + 55039202 55039221 GATTGATTCAGAAGTCTCAC 1092 −327 gRNA058 + 55039210 55039229 CAGAAGTCTCACTGGTCAGC 1093 −319 gRNA059 + 55039218 55039237 TCACTGGTCAGCAGGAGACA 1094 −311 gRNA060 + 55039228 55039247 GCAGGAGACAAGGTGGACCC 1095 −301 gRNA061 + 55039242 55039261 GGACCCAGGAAACACTGAAA 1096 −287 gRNA062 + 55039245 55039264 CCCAGGAAACACTGAAAAGG 1097 −284 gRNA063 + 55039246 55039265 CCAGGAAACACTGAAAAGGT 1098 −283 gRNA064 + 55039251 55039270 AAACACTGAAAAGGTGGGCC 1099 −278 gRNA065 + 55039281 55039300 TGGAGTCTGGCATCCCACGC 1100 −248 gRNA066 + 55039282 55039301 GGAGTCTGGCATCCCACGCA 1101 −247 gRNA067 + 55039312 55039331 CGGGAGAGGAGGAGCCCCTA 1102 −217 gRNA068 + 55039318 55039337 AGGAGGAGCCCCTAGGGCGC 1103 −211 gRNA069 − 55039329 55039348 AAGGCAGGCCGGCGCCCTAG 1104 −200 gRNA070 − 55039330 55039349 GAAGGCAGGCCGGCGCCCTA 1105 −199 gRNA071 − 55039331 55039350 GGAAGGCAGGCCGGCGCCCT 1106 −198 gRNA072 − 55039340 55039359 AACTGGGCTGGAAGGCAGGC 1107 −189 gRNA073 + 55039340 55039359 GCCTGCCTTCCAGCCCAGTT 1108 −189 gRNA074 − 55039344 55039363 TCCTAACTGGGCTGGAAGGC 1109 −185 gRNA075 + 55039346 55039365 CTTCCAGCCCAGTTAGGATT 1110 −183 gRNA076 + 55039347 55039366 TTCCAGCCCAGTTAGGATTT 1111 −182 gRNA077 − 55039348 55039367 CAAATCCTAACTGGGCTGGA 1112 −181 gRNA078 − 55039352 55039371 CTCCCAAATCCTAACTGGGC 1113 −177 gRNA079 − 55039356 55039375 AAAACTCCCAAATCCTAACT 1114 −173 gRNA080 − 55039357 55039376 AAAAACTCCCAAATCCTAAC 1115 −172 gRNA081 − 55039384 55039403 AGCGTCAGATTACGCGCAGA 1116 −145 gRNA082 − 55039385 55039404 CAGCGTCAGATTACGCGCAG 1117 −144 gRNA083 + 55039387 55039406 GCGCGTAATCTGACGCTGTT 1118 −142 gRNA084 + 55039388 55039407 CGCGTAATCTGACGCTGTTT 1119 −141 gRNA085 + 55039389 55039408 GCGTAATCTGACGCTGTTTG 1120 −140 gRNA086 + 55039392 55039411 TAATCTGACGCTGTTTGGGG 1121 −137 gRNA087 + 55039393 55039412 AATCTGACGCTGTTTGGGGA 1122 −136 gRNA088 + 55039398 55039417 GACGCTGTTTGGGGAGGGCG 1123 −131 gRNA089 + 55039422 55039441 CGAAACCTGATCCTCCAGTC 1124 −107 gRNA090 + 55039423 55039442 GAAACCTGATCCTCCAGTCC 1125 −106 gRNA091 + 55039424 55039443 AAACCTGATCCTCCAGTCCG 1126 −105 gRNA092 − 55039424 55039443 CGGACTGGAGGATCAGGTTT 1127 −105 gRNA093 + 55039425 55039444 AACCTGATCCTCCAGTCCGG 1128 −104 gRNA094 − 55039430 55039449 AACCCCCGGACTGGAGGATC 1129 −99 gRNA095 − 55039436 55039455 TAACGGAACCCCCGGACTGG 1130 −93 gRNA096 − 55039439 55039458 CATTAACGGAACCCCCGGAC 1131 −90 gRNA097 − 55039444 55039463 TTAAACATTAACGGAACCCC 1132 −85 gRNA098 + 55039450 55039469 CCGTTAATGTTTAATCAGAT 1133 −79 gRNA099 − 55039453 55039472 CCTATCTGATTAAACATTAA 1134 −76 gRNA100 + 55039462 55039481 AATCAGATAGGATCGTCCGA 1135 −67 gRNA101 + 55039463 55039482 ATCAGATAGGATCGTCCGAT 1136 −66 gRNA102 + 55039493 55039512 TGGCGTGATCTGCGCGCCCC 1137 −36 gRNA103 − 55039534 55039553 GTCGCTGCGGAAACCTTCTA 1138 5 gRNA104 − 55039535 55039554 CGTCGCTGCGGAAACCTTCT 1139 6 gRNA105 + 55039538 55039557 AGGTTTCCGCAGCGACGTCG 1140 9 gRNA106 + 55039547 55039566 CAGCGACGTCGAGGCGCTCA 1141 18 gRNA107 − 55039547 55039566 TGAGCGCCTCGACGTCGCTG 1142 18 gRNA108 + 55039554 55039573 GTCGAGGCGCTCATGGTTGC 1143 25 gRNA109 + 55039557 55039576 GAGGCGCTCATGGTTGCAGG 1144 28 gRNA110 + 55039558 55039577 AGGCGCTCATGGTTGCAGGC 1145 29 gRNA111 + 55039592 55039611 AGTTCAGGGTCTGAGCCTGG 1146 63 gRNA112 + 55039577 55039596 CGGGCGCCGCCGTTCAGTTC 1147 48 gRNA113 + 55039578 55039597 GGGCGCCGCCGTTCAGTTCA 1148 49 gRNA114 − 55039586 55039605 TCAGACCCTGAACTGAACGG 1149 57 gRNA115 − 55039589 55039608 GGCTCAGACCCTGAACTGAA 1150 60 gRNA116 + 55039589 55039608 TTCAGTTCAGGGTCTGAGCC 1151 60 gRNA117 + 55039603 55039622 TGAGCCTGGAGGAGTGAGCC 1152 74 gRNA118 + 55039615 55039634 AGTGAGCCAGGCAGTGAGAC 1153 86 gRNA119 + 55039620 55039639 GCCAGGCAGTGAGACTGGCT 1154 91 gRNA120 + 55039621 55039640 CCAGGCAGTGAGACTGGCTC 1155 92 gRNA121 − 55039624 55039643 CCCGAGCCAGTCTCACTGCC 1156 95 gRNA122 + 55039624 55039643 GGCAGTGAGACTGGCTCGGG 1157 95 gRNA123 + 55039625 55039644 GCAGTGAGACTGGCTCGGGC 1158 96 gRNA124 + 55039629 55039648 TGAGACTGGCTCGGGCGGGC 1159 100 gRNA125 + 55039630 55039649 GAGACTGGCTCGGGCGGGCC 1160 101 gRNA126 + 55039650 55039669 GGGACGCGTCGTTGCAGCAG 1161 121 gRNA127 − 55039651 55039670 GCTGCTGCAACGACGCGTCC 1162 122 gRNA128 − 55039690 55039709 TGAAGGGGCGCGCGGAATCC 1163 161 gRNA129 − 55039698 55039717 AGGGCGCGTGAAGGGGCGCG 1164 169 gRNA130 − 55039705 55039724 CAGGAGCAGGGCGCGTGAAG 1165 176 gRNA131 − 55039706 55039725 TCAGGAGCAGGGCGCGTGAA 1166 177 gRNA132 − 55039707 55039726 TTCAGGAGCAGGGCGCGTGA 1167 178 gRNA133 − 55039717 55039736 GGAGCTGAAGTTCAGGAGCA 1168 188 gRNA134 − 55039718 55039737 AGGAGCTGAAGTTCAGGAGC 1169 189 gRNA135 − 55039724 55039743 CTGTGCAGGAGCTGAAGTTC 1170 195 gRNA136 + 55039738 55039757 GCACAGTCCTCCCCACCGCA 1171 209 gRNA137 − 55039738 55039757 TGCGGTGGGGAGGACTGTGC 1172 209 gRNA138 + 55039745 55039764 CCTCCCCACCGCAAGGCTCA 1173 216 gRNA139 − 55039748 55039767 CCTTGAGCCTTGCGGTGGGG 1174 219 gRNA140 − 55039751 55039770 GCGCCTTGAGCCTTGCGGTG 1175 222 gRNA141 − 55039752 55039771 GGCGCCTTGAGCCTTGCGGT 1176 223 gRNA142 − 55039753 55039772 CGGCGCCTTGAGCCTTGCGG 1177 224 gRNA143 + 55039754 55039773 CGCAAGGCTCAAGGCGCCGC 1178 225 gRNA144 + 55039759 55039778 GGCTCAAGGCGCCGCCGGCG 1179 230 gRNA145 − 55039776 55039795 AGGCCGTGCGCGGTCCACGC 1180 247 gRNA146 + 55039778 55039797 GTGGACCGCGCACGGCCTCT 1181 249 gRNA147 − 55039786 55039805 GGAGACCTAGAGGCCGTGCG 1182 257 gRNA148 + 55039810 55039829 CAGGACAGCAACCTCTCCCC 1183 281 gRNA149 + 55039839 55039858 TGGGCACCGTCAGCTCCAGG 1184 310 gRNA150 + 55039845 55039864 CCGTCAGCTCCAGGCGGTCC 1185 316 gRNA151 − 55039848 55039867 CCAGGACCGCCTGGAGCTGA 1186 319 gRNA152 + 55039848 55039867 TCAGCTCCAGGCGGTCCTGG 1187 319 gRNA153 − 55039857 55039876 CAGCGGCCACCAGGACCGCC 1188 328 gRNA154 + 55039895 55039914 CTGCTGCTCCTGGGTCCCGC 1189 366 gRNA155 − 55039906 55039925 CACGGGCGCCCGCGGGACCC 1190 377 gRNA156 + 55039909 55039928 TCCCGCGGGCGCCCGTGCGC 1191 380 gRNA157 + 55039912 55039931 CGCGGGCGCCCGTGCGCAGG 1192 383 gRNA158 − 55039913 55039932 TCCTGCGCACGGGCGCCCGC 1193 384 gRNA159 − 55039914 55039933 CTCCTGCGCACGGGCGCCCG 1194 385 gRNA160 + 55039918 55039937 CGCCCGTGCGCAGGAGGACG 1195 389 gRNA161 + 55039922 55039941 CGTGCGCAGGAGGACGAGGA 1196 393 gRNA162 − 55039923 55039942 GTCCTCGTCCTCCTGCGCAC 1197 394 gRNA163 − 55039924 55039943 CGTCCTCGTCCTCCTGCGCA 1198 395 gRNA164 + 55039933 55039952 GGACGAGGACGGCGACTACG 1199 404 gRNA165 + 55039939 55039958 GGACGGCGACTACGAGGAGC 1200 410 gRNA166 + 55039963 55039982 GCTAGCCTTGCGTTCCGAGG 1201 434 gRNA167 + 55039967 55039986 GCCTTGCGTTCCGAGGAGGA 1202 438 gRNA168 − 55039971 55039990 GCCGTCCTCCTCGGAACGCA 1203 442 gRNA169 + 55039972 55039991 GCGTTCCGAGGAGGACGGCC 1204 443 gRNA170 − 55039980 55039999 TTCGGCCAGGCCGTCCTCCT 1205 451 gRNA171 − 55039993 55040012 CGTGCTCGGGTGCTTCGGCC 1206 464 gRNA172 − 55040006 55040025 GTGGCTGTGGTTCCGTGCTC 1207 477 gRNA173 − 55040007 55040026 GGTGGCTGTGGTTCCGTGCT 1208 478 gRNA174 − 55040019 55040038 GCAGCGGTGGAAGGTGGCTG 1209 490 gRNA175 + 55040023 55040042 CACCTTCCACCGCTGCGCCA 1210 494 gRNA176 − 55040025 55040044 CTTGGCGCAGCGGTGGAAGG 1211 496 gRNA177 − 55040028 55040047 CACCTTGGCGCAGCGGTGGA 1212 499 gRNA178 + 55040028 55040047 TCCACCGCTGCGCCAAGGTG 1213 499 gRNA179 + 55040029 55040048 CCACCGCTGCGCCAAGGTGC 1214 500 gRNA180 − 55040032 55040051 CCCGCACCTTGGCGCAGCGG 1215 503 gRNA181 + 55040070 55040089 GGGCGAACCCGCAGCCGGGA 1216 541 gRNA182 − 55040070 55040089 TCCCGGCTGCGGGTTCGCCC 1217 541 gRNA183 − 55040080 55040099 CACCGCACCGTCCCGGCTGC 1218 551 gRNA184 − 55040081 55040100 GCACCGCACCGTCCCGGCTG 1219 552 gRNA185 − 55040087 55040106 GAAACAGCACCGCACCGTCC 1220 558 gRNA186 + 55040091 55040110 GGTGCGGTGCTGTTTCCTCT 1221 562 gRNA187 + 55040092 55040111 GTGCGGTGCTGTTTCCTCTC 1222 563 gRNA188 − 55040109 55040128 GGGGGAAACTGAGGCCCGAG 1223 580 gRNA189 + 55040119 55040138 AGTTTCCCCCCATGTAAGAG 1224 590 gRNA190 + 55040125 55040144 CCCCCATGTAAGAGAGGAAG 1225 596 gRNA191 − 55040127 55040146 CACTTCCTCTCTTACATGGG 1226 598 gRNA192 − 55040128 55040147 CCACTTCCTCTCTTACATGG 1227 599 gRNA193 + 55040133 55040152 TAAGAGAGGAAGTGGAGTGC 1228 604 gRNA194 + 55040155 55040174 GTCGCCGAGGGCTCTTCGCT 1229 626 gRNA195 − 55040162 55040181 CGTGCCAAGCGAAGAGCCCT 1230 633 gRNA196 + 55040166 55040185 CTCTTCGCTTGGCACGATCT 1231 637 gRNA197 + 55040167 55040186 TCTTCGCTTGGCACGATCTT 1232 638 gRNA198 + 55040168 55040187 CTTCGCTTGGCACGATCTTG 1233 639 gRNA199 + 55040176 55040195 GGCACGATCTTGGGGACTGC 1234 647 gRNA200 + 55040181 55040200 GATCTTGGGGACTGCAGGCA 1235 652 gRNA201 + 55040184 55040203 CTTGGGGACTGCAGGCAAGG 1236 655 gRNA202 + 55040189 55040208 GGACTGCAGGCAAGGCGGCG 1237 660 gRNA203 + 55040190 55040209 GACTGCAGGCAAGGCGGCGG 1238 661 gRNA204 + 55040193 55040212 TGCAGGCAAGGCGGCGGGGG 1239 664 gRNA205 + 55040197 55040216 GGCAAGGCGGCGGGGGAGGA 1240 668 gRNA206 + 55040198 55040217 GCAAGGCGGCGGGGGAGGAC 1241 669 gRNA207 + 55040223 55040242 GTGGGGAGCACGGTGGAGAG 1242 694 gRNA208 + 55040224 55040243 TGGGGAGCACGGTGGAGAGC 1243 695 gRNA209 + 55040225 55040244 GGGGAGCACGGTGGAGAGCG 1244 696 gRNA210 + 55040229 55040248 AGCACGGTGGAGAGCGGGGA 1245 700 gRNA211 + 55040233 55040252 CGGTGGAGAGCGGGGACGGC 1246 704 gRNA212 + 55040277 55040296 CGTGCGGCTGCGCTATTCAG 1247 748 gRNA213 + 55040278 55040297 GTGCGGCTGCGCTATTCAGT 1248 749 gRNA214 + 55040282 55040301 GGCTGCGCTATTCAGTGGGA 1249 753 gRNA215 + 55040289 55040308 CTATTCAGTGGGAAGGTTCG 1250 760 gRNA216 + 55040290 55040309 TATTCAGTGGGAAGGTTCGC 1251 761 gRNA217 + 55040291 55040310 ATTCAGTGGGAAGGTTCGCG 1252 762 gRNA218 + 55040295 55040314 AGTGGGAAGGTTCGCGGGGT 1253 766 gRNA219 + 55040296 55040315 GTGGGAAGGTTCGCGGGGTT 1254 767 gRNA220 + 55040337 55040356 AGGGCGAGCAGAGCACTGCC 1255 808 gRNA221 + 55040382 55040401 TTTCTGCCTCGCCGCGGCAC 1256 853 gRNA222 + 55040385 55040404 CTGCCTCGCCGCGGCACAGG 1257 856 gRNA223 + 55040386 55040405 TGCCTCGCCGCGGCACAGGT 1258 857 gRNA224 − 55040391 55040410 CACCCACCTGTGCCGCGGCG 1259 862 gRNA225 − 55040396 55040415 TCCTTCACCCACCTGTGCCG 1260 867 gRNA226 + 55040405 55040424 TGGGTGAAGGAGTGAATGCC 1261 876

In some embodiments, the gRNA herein does not comprise the sequence

(SEQ ID NO: 1490) CCCGCACCUUGGCGCAGCGG.

Any tracr sequence known in the art is contemplated for a gRNA described herein. In some embodiments, a gRNA described herein has a tracr sequence shown in Table 3 below, or a tracr sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the tracr sequence shown below (SEQ: SEQ ID NO).

TABLE 3 Exemplary TRACR Sequences SEQ Sequence (5′ to 3′) 653 GUUUAAGAGCUAUGCUGGAAACAGCAUAGC AAGUUUAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGCUUUU UUU 654 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGCUUUU 655 GUUUAAGAGCUAAGCUGGAAACAGCAUAGC AAGUUUAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGCUUUU UU 656 GUUUAAGAGCUAAGCUGGAAACAGCAUAGC AAGUUUAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGCUUUU UUU

In some embodiments, the gRNA herein is provided to the cell directly (e.g., through an RNP complex together with the CRISPR-associated protein domain). In some embodiments, the gRNA is provided to the cell through an expression vector (e.g., a plasmid vector or a viral vector) introduced into the cell, where the cell then expresses the gRNA from the expression vector. Methods of introducing gRNAs and expression vectors into cells are well known in the art.

III. Effector Domains

Epigenetic editors described herein include one or more effector protein domains (also “epigenetic effector domains,” or “effector domains,” as used herein) that effect epigenetic modification of a target gene. An epigenetic editor with one or more effector domains may modulate expression of a target gene without altering its nucleobase sequence. In some embodiments, an effector domain described herein may provide repression or silencing of expression of a target gene such as PCSK9, e.g., by repressing transcription or by modifying or remodeling chromatin. Such effector domains are also referred to herein as “repression domains,” “repressor domains,” or “epigenetic repressor domains.” Non-limiting examples of chemical modifications that may be mediated by effector domains include methylation, demethylation, acetylation, deacetylation, phosphorylation, SUMOylation and/or ubiquitination of DNA or histone residues.

In some embodiments, an effector domain of an epigenetic editor described herein may make histone tail modifications, e.g., by adding or removing active marks on histone tails.

In some embodiments, an effector domain of an epigenetic editor described herein may comprise or recruit a transcription-related protein, e.g., a transcription repressor. The transcription-related protein may be endogenous or exogenous.

In some embodiments, an effector domain of an epigenetic editor described herein may, for example, comprise a protein that directly or indirectly blocks access of a transcription factor to the gene of interest harboring the target sequence.

An effector domain may be a full-length protein or a fragment thereof that retains the epigenetic effector function (a “functional domain”). Functional domains that are capable of modulating (e.g., repressing) gene expression can be derived from a larger protein. For example, functional domains that can reduce target gene expression may be identified based on sequences of repressor proteins. Amino acid sequences of gene expression-modulating proteins may be obtained from available genome browsers, such as the UCSD genome browser or Ensembl genome browser. Protein annotation databases such as UniProt or Pfam can be used to identify functional domains within the full protein sequence. As a starting point, the largest sequence, encompassing all regions identified by different databases, may be tested for gene expression modulation activity. Various truncations then may be tested to identify the minimal functional unit.

Variants of effector domains described herein are also contemplated by the present disclosure. A variant may, for example, refer to a polypeptide with at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence similarity to a wildtype effector domain described herein. In particular embodiments, the variant retains at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the epigenetic effector function of the wildtype effector domain.

In some embodiments, an effector domain described herein may comprise a fusion of two or more effector domains (e.g., KOX1 KRAB and ZIM3). The effector domain may, for example, comprise a fusion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 effector domains, such as effector domains described herein. In certain embodiments, an effector domain comprises a fusion of a truncated form of an effector domain and a second effector domain. In certain embodiments, an effector domain comprises a fusion of the truncated forms of two effector domains (e.g., fusions of the N- and C-terminal portions of the two effector domains).

In some embodiments, an epigenetic editor described herein may comprise 1 effector domain, 2 effector domains, 3 effector domains, 4 effector domains, 5 effector domains, 6 effector domains, 7 effector domains, 8 effector domains, 9 effector domains, 10 effector domains, or more. In certain embodiments, the epigenetic editor comprises one or more fusion proteins (e.g., one, two, or three fusion proteins), each with one or more effector domains (e.g., one, two, or three effector domains) linked to a DNA-binding domain. In some embodiments, the effector domains may induce a combination of epigenetic modifications, e.g., transcription repression and DNA methylation, DNA methylation and histone deacetylation, DNA methylation and histone demethylation, DNA methylation and histone methylation, DNA methylation and histone phosphorylation, DNA methylation and histone ubiquitylation, DNA methylation, and histone SUMOylation.

In certain embodiments, an effector domain described herein (e.g., DNMT3A and/or DNMT3L) is encoded by a nucleotide sequence as found in the native genome (e.g., human or murine) for that effector domain. In other embodiments, an effector domain described herein is encoded by a nucleotide sequence that has been codon-optimized for optimal expression in human cells.

Effector domains described herein may include, for example, transcriptional repressors, DNA methyltransferases, and/or histone modifiers, as further detailed below.

A. Transcriptional Repressors

In some embodiments, an epigenetic effector domain described herein mediates repression of a target gene's expression (e.g., transcription). The effector domain may comprise, e.g., a Kruppel-associated box (KRAB) repressor domain, a Repressor Element Silencing Transcription Factor (REST) repressor domain, a KRAB-associated protein 1 (KAP1) domain, a MAD domain, a FKHR (forkhead in rhabdosarcoma gene) repressor domain, an EGR-1 (early growth response gene product-1) repressor domain, an ets2 repressor factor repressor domain (ERD), a MAD smSIN3 interaction domain (SID), a WRPW motif of the hairy-related basic helix-loop-helix (bHLH) repressor proteins, an HP1 alpha chromo-shadow repressor domain, an HP1 beta repressor domain, or any combination thereof. The effector domain may recruit one or more protein domains that repress expression of the target gene, e.g., through a scaffold protein. In some embodiments, the effector domain may recruit or interact with a scaffold protein domain that recruits a PRMT protein, a HDAC protein, a SETDB1 protein, or a NuRD protein domain.

In some embodiments, the effector domain comprises a functional domain derived from a zinc finger repressor protein, such as a KRAB domain. KRAB domains are found in approximately 400 human ZFP-based transcription factors. Descriptions of KRAB domains may be found, for example, in Ecco et al., Development (2017) 144(15):2719-29 and Lambert et al., Cell (2018) 172:650-65.

In certain embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from KOX1/ZNF10, KOX8/ZNF708, ZNF43, ZNF184, ZNF91, HPF4, HTF10, or HTF34. In some embodiments, the effector domain comprises a repressor domain (e.g., KRAB) derived from ZIM3, ZNF436, ZNF257, ZNF675, ZNF490, ZNF320, ZNF331, ZNF816, ZNF680, ZNF41, ZNF189, ZNF528, ZNF543, ZNF554, ZNF140, ZNF610, ZNF264, ZNF350, ZNF8, ZNF582, ZNF30, ZNF324, ZNF98, ZNF669, ZNF677, ZNF596, ZNF214, ZNF37, ZNF34, ZNF250, ZNF547, ZNF273, ZNF354, ZFP82, ZNF224, ZNF33, ZNF45, ZNF175, ZNF595, ZNF184, ZNF419, ZFP28-1, ZFP28-2, ZNF18, ZNF213, ZNF394, ZFP1, ZFP14, ZNF416, ZNF557, ZNF566, ZNF729, ZIM2, ZNF254, ZNF764, ZNF785, or any combination thereof. For example, the repressor domain may be a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627. In particular embodiments, the repressor domain is a ZIM3 KRAB domain. In further embodiments, the effector domain is derived from a human protein, e.g., a human ZIM3, a human KOX1, a human ZFP28, or a human ZN627.

Sequences of exemplary effector domains that may reduce or silence target gene expression, or protein sequences that contain them, are provided in Table 4 below (SEQ: SEQ ID NO). Further examples of repressors and transcriptional repressor domains can be found, e.g., in PCT Patent Publication WO 2021/226077 and Tycko et al., Cell (2020) 183(7):2020-35, each of which is incorporated herein by reference in its entirety.

TABLE 4 Exemplary Effector Domains That May Reduce or Silence Gene Expression Protein SEQ ZIM3 33 ZNF436 34 ZNF257 35 ZNF675 36 ZNF490 37 ZNF320 38 ZNF331 39 ZNF816 40 ZNF680 41 ZNF41 42 ZNF189 43 ZNF528 44 ZNF543 45 ZNF554 46 ZNF140 47 ZNF610 48 ZNF264 49 ZNF350 50 ZNF8 51 ZNF582 52 ZNF30 53 ZNF324 54 ZNF98 55 ZNF669 56 ZNF677 57 ZNF596 58 ZNF214 59 ZNF37A 60 ZNF34 61 ZNF250 62 ZNF547 63 ZNF273 64 ZNF354A 65 ZFP82 66 ZNF224 67 ZNF33A 68 ZNF45 69 ZNF175 70 ZNF595 71 ZNF184 72 ZNF419 73 ZFP28-1 74 ZFP28-2 75 ZNF18 76 ZNF213 77 ZNF394 78 ZFP1 79 ZFP14 80 ZNF416 81 ZNF557 82 ZNF566 83 ZNF729 84 ZIM2 85 ZNF254 86 ZNF764 87 ZNF785 88 ZNF10 (KOX1) 89 CBX5 (chromoshadow domain) 90 RYBP (YAF2_RYBP 91 component of PRC1) YAF2 (YAF2_RYBP 92 component of PRC1) MGA (component of PRC1.6) 93 CBX1 (chromoshadow) 94 SCMH1 (SAM_1/SPM) 95 MPP8 (Chromodomain) 96 SUMO3 (Rad60-SLD) 97 HERC2 (Cyt-b5) 98 BIN1 (SH3_9) 99 PCGF2 (RING finger protein 100 domain) TOX (HMG box) 101 FOXA1 (HNF3A C-terminal 102 domain) FOXA2 (HNF3B C-terminal 103 domain) IRF2BP1 (IRF-2BP1_2 N- 104 terminal domain) IRF2BP2 (IRF-2BP1_2 N- 105 terminal domain) IRF2BPL IRF-2BP1_2 N- 106 terminal domain HOXA13 (homeodomain) 107 HOXB13 (homeodomain) 108 HOXC13 (homeodomain) 109 HOXA11 (homeodomain) 110 HOXC11 (homeodomain) 111 HOXC10 (homeodomain) 112 HOXA10 (homeodomain) 113 HOXB9 (homeodomain) 114 HOXA9 (homeodomain) 115 ZFP28_HUMAN 116 ZN334_HUMAN 117 ZN568_HUMAN 118 ZN37A_HUMAN 119 ZN181_HUMAN 120 ZN510_HUMAN 121 ZN862_HUMAN 122 ZN140_HUMAN 123 ZN208_HUMAN 124 ZN248_HUMAN 125 ZN571_HUMAN 126 ZN699_HUMAN 127 ZN726_HUMAN 128 ZIK1_HUMAN 129 ZNF2_HUMAN 130 Z705F_HUMAN 131 ZNF14_HUMAN 132 ZN471_HUMAN 133 ZN624_HUMAN 134 ZNF84_HUMAN 135 ZNF7_HUMAN 136 ZN891_HUMAN 137 ZN337_HUMAN 138 Z705G_HUMAN 139 ZN529_HUMAN 140 ZN729_HUMAN 141 ZN419_HUMAN 142 Z705A_HUMAN 143 ZNF45_HUMAN 144 ZN302_HUMAN 145 ZN486_HUMAN 146 ZN621_HUMAN 147 ZN688_HUMAN 148 ZN33A_HUMAN 149 ZN554_HUMAN 150 ZN878_HUMAN 151 ZN772_HUMAN 152 ZN224_HUMAN 153 ZN184_HUMAN 154 ZN544_HUMAN 155 ZNF57_HUMAN 156 ZN283_HUMAN 157 ZN549_HUMAN 158 ZN211_HUMAN 159 ZN615_HUMAN 160 ZN253_HUMAN 161 ZN226_HUMAN 162 ZN730_HUMAN 163 Z585A_HUMAN 164 ZN732_HUMAN 165 ZN681_HUMAN 166 ZN667_HUMAN 167 ZN649_HUMAN 168 ZN470_HUMAN 169 ZN484_HUMAN 170 ZN431_HUMAN 171 ZN382_HUMAN 172 ZN254_HUMAN 173 ZN124_HUMAN 174 ZN607_HUMAN 175 ZN317_HUMAN 176 ZN620_HUMAN 177 ZN141_HUMAN 178 ZN584_HUMAN 179 ZN540_HUMAN 180 ZN75D_HUMAN 181 ZN555_HUMAN 182 ZN658_HUMAN 183 ZN684_HUMAN 184 RBAK_HUMAN 185 ZN829_HUMAN 186 ZN582_HUMAN 187 ZN112_HUMAN 188 ZN716_HUMAN 189 HKR1_HUMAN 190 ZN350_HUMAN 191 ZN480_HUMAN 192 ZN416_HUMAN 193 ZNF92_HUMAN 194 ZN100_HUMAN 195 ZN736_HUMAN 196 ZNF74_HUMAN 197 CBX1_HUMAN 198 ZN443_HUMAN 199 ZN195_HUMAN 200 ZN530_HUMAN 201 ZN782_HUMAN 202 ZN791_HUMAN 203 ZN331_HUMAN 204 Z354C_HUMAN 205 ZN157_HUMAN 206 ZN727_HUMAN 207 ZN550_HUMAN 208 ZN793_HUMAN 209 ZN235_HUMAN 210 ZNF8_HUMAN 211 ZN724_HUMAN 212 ZN573_HUMAN 213 ZN577_HUMAN 214 ZN789_HUMAN 215 ZN718_HUMAN 216 ZN300_HUMAN 217 ZN383_HUMAN 218 ZN429_HUMAN 219 ZN677_HUMAN 220 ZN850_HUMAN 221 ZN454_HUMAN 222 ZN257_HUMAN 223 ZN264_HUMAN 224 ZFP82_HUMAN 225 ZFP14_HUMAN 226 ZN485_HUMAN 227 ZN737_HUMAN 228 ZNF44_HUMAN 229 ZN596_HUMAN 230 ZN565_HUMAN 231 ZN543_HUMAN 232 ZFP69_HUMAN 233 SUMO1_HUMAN 234 ZNF12_HUMAN 235 ZN169_HUMAN 236 ZN433_HUMAN 237 SUMO3_HUMAN 238 ZNF98_HUMAN 239 ZN175_HUMAN 240 ZN347_HUMAN 241 ZNF25_HUMAN 242 ZN519_HUMAN 243 Z585B_HUMAN 244 ZIM3_HUMAN 245 ZN517_HUMAN 246 ZN846_HUMAN 247 ZN230_HUMAN 248 ZNF66_HUMAN 249 ZFP1_HUMAN 250 ZN713_HUMAN 251 ZN816_HUMAN 252 ZN426_HUMAN 253 ZN674_HUMAN 254 ZN627_HUMAN 255 ZNF20_HUMAN 256 Z587B_HUMAN 257 ZN316_HUMAN 258 ZN233_HUMAN 259 ZN611_HUMAN 260 ZN556_HUMAN 261 ZN234_HUMAN 262 ZN560_HUMAN 263 ZNF77_HUMAN 264 ZN682_HUMAN 265 ZN614_HUMAN 266 ZN785_HUMAN 267 ZN445_HUMAN 268 ZFP30_HUMAN 269 ZN225_HUMAN 270 ZN551_HUMAN 271 ZN610_HUMAN 272 ZN528_HUMAN 273 ZN284_HUMAN 274 ZN418_HUMAN 275 MPP8_HUMAN 276 ZN490_HUMAN 277 ZN805_HUMAN 278 Z780B_HUMAN 279 ZN763_HUMAN 280 ZN285_HUMAN 281 ZNF85_HUMAN 282 ZN223_HUMAN 283 ZNF90_HUMAN 284 ZN557_HUMAN 285 ZN425_HUMAN 286 ZN229_HUMAN 287 ZN606_HUMAN 288 ZN155_HUMAN 289 ZN222_HUMAN 290 ZN442_HUMAN 291 ZNF91_HUMAN 292 ZN135_HUMAN 293 ZN778_HUMAN 294 RYBP_HUMAN 295 ZN534_HUMAN 296 ZN586_HUMAN 297 ZN567_HUMAN 298 ZN440_HUMAN 299 ZN583_HUMAN 300 ZN441_HUMAN 301 ZNF43_HUMAN 302 CBX5_HUMAN 303 ZN589_HUMAN 304 ZNF10_HUMAN 305 ZN563_HUMAN 306 ZN561_HUMAN 307 ZN136_HUMAN 308 ZN630_HUMAN 309 ZN527_HUMAN 310 ZN333_HUMAN 311 Z324B_HUMAN 312 ZN786_HUMAN 313 ZN709_HUMAN 314 ZN792_HUMAN 315 ZN599_HUMAN 316 ZN613_HUMAN 317 ZF69B_HUMAN 318 ZN799_HUMAN 319 ZN569_HUMAN 320 ZN564_HUMAN 321 ZN546_HUMAN 322 ZFP92_HUMAN 323 YAF2_HUMAN 324 ZN723_HUMAN 325 ZNF34_HUMAN 326 ZN439_HUMAN 327 ZFP57_HUMAN 328 ZNF19_HUMAN 329 ZN404_HUMAN 330 ZN274_HUMAN 331 CBX3_HUMAN 332 ZNF30_HUMAN 333 ZN250_HUMAN 334 ZN570_HUMAN 335 ZN675_HUMAN 336 ZN695_HUMAN 337 ZN548_HUMAN 338 ZN132_HUMAN 339 ZN738_HUMAN 340 ZN420_HUMAN 341 ZN626_HUMAN 342 ZN559_HUMAN 343 ZN460_HUMAN 344 ZN268_HUMAN 345 ZN304_HUMAN 346 ZIM2_HUMAN 347 ZN605_HUMAN 348 ZN844_HUMAN 349 SUMO5_HUMAN 350 ZN101_HUMAN 351 ZN783_HUMAN 352 ZN417_HUMAN 353 ZN182_HUMAN 354 ZN823_HUMAN 355 ZN177_HUMAN 356 ZN197_HUMAN 357 ZN717_HUMAN 358 ZN669_HUMAN 359 ZN256_HUMAN 360 ZN251_HUMAN 361 CBX4_HUMAN 362 PCGF2_HUMAN 363 CDY2_HUMAN 364 CDYL2_HUMAN 365 HERC2_HUMAN 366 ZN562_HUMAN 367 ZN461_HUMAN 368 Z324A_HUMAN 369 ZN766_HUMAN 370 ID2_HUMAN 371 TOX_HUMAN 372 ZN274_HUMAN 373 SCMH1_HUMAN 374 ZN214_HUMAN 375 CBX7_HUMAN 376 ID1_HUMAN 377 CREM_HUMAN 378 SCX_HUMAN 379 ASCL1_HUMAN 380 ZN764_HUMAN 381 SCML2_HUMAN 382 TWST1_HUMAN 383 CREB1_HUMAN 384 TERF1_HUMAN 385 ID3_HUMAN 386 CBX8_HUMAN 387 CBX4_HUMAN 388 GSX1_HUMAN 389 NKX22_HUMAN 390 ATF1_HUMAN 391 TWST2_HUMAN 392 ZNF17_HUMAN 393 TOX3_HUMAN 394 TOX4_HUMAN 395 ZMYM3_HUMAN 396 I2BP1_HUMAN 397 RHXF1_HUMAN 398 SSX2_HUMAN 399 I2BPL_HUMAN 400 ZN680_HUMAN 401 CBX1_HUMAN 402 TRI68_HUMAN 403 HXA13_HUMAN 404 PHC3_HUMAN 405 TCF24_HUMAN 406 CBX3_HUMAN 407 HXB13_HUMAN 408 HEY1_HUMAN 409 PHC2_HUMAN 410 ZNF81_HUMAN 411 FIGLA_HUMAN 412 SAM11_HUMAN 413 KMT2B_HUMAN 414 HEY2_HUMAN 415 JDP2_HUMAN 416 HXC13_HUMAN 417 ASCL4_HUMAN 418 HHEX_HUMAN 419 HERC2_HUMAN 420 GSX2_HUMAN 421 BIN1_HUMAN 422 ETV7_HUMAN 423 ASCL3_HUMAN 424 PHC1_HUMAN 425 OTP_HUMAN 426 I2BP2_HUMAN 427 VGLL2_HUMAN 428 HXA11_HUMAN 429 PDLI4_HUMAN 430 ASCL2_HUMAN 431 CDX4_HUMAN 432 ZN860_HUMAN 433 LMBL4_HUMAN 434 PDIP3_HUMAN 435 NKX25_HUMAN 436 CEBPB_HUMAN 437 ISL1_HUMAN 438 CDX2_HUMAN 439 PROP1_HUMAN 440 SIN3B_HUMAN 441 SMBT1_HUMAN 442 HXC11_HUMAN 443 HXC10_HUMAN 444 PRS6A_HUMAN 445 VSX1_HUMAN 446 NKX23_HUMAN 447 MTG16_HUMAN 448 HMX3_HUMAN 449 HMX1_HUMAN 450 KIF22_HUMAN 451 CSTF2_HUMAN 452 CEBPE_HUMAN 453 DLX2_HUMAN 454 ZMYM3_HUMAN 455 PPARG_HUMAN 456 PRIC1_HUMAN 457 UNC4_HUMAN 458 BARX2_HUMAN 459 ALX3_HUMAN 460 TCF15_HUMAN 461 TERA_HUMAN 462 VSX2_HUMAN 463 HXD12_HUMAN 464 CDX1_HUMAN 465 TCF23_HUMAN 466 ALX1_HUMAN 467 HXA10_HUMAN 468 RX_HUMAN 469 CXXC5_HUMAN 470 SCML1_HUMAN 471 NFIL3_HUMAN 472 DLX6_HUMAN 473 MTG8_HUMAN 474 CBX8_HUMAN 475 CEBPD_HUMAN 476 SEC13_HUMAN 477 FIP1_HUMAN 478 ALX4_HUMAN 479 LHX3_HUMAN 480 PRIC2_HUMAN 481 MAGI3_HUMAN 482 NELL1_HUMAN 483 PRRX1_HUMAN 484 MTG8R_HUMAN 485 RAX2_HUMAN 486 DLX3_HUMAN 487 DLX1_HUMAN 488 NKX26_HUMAN 489 NAB1_HUMAN 490 SAMD7_HUMAN 491 PITX3_HUMAN 492 WDR5_HUMAN 493 MEOX2_HUMAN 494 NAB2_HUMAN 495 DHX8_HUMAN 496 FOXA2_HUMAN 497 CBX6_HUMAN 498 EMX2_HUMAN 499 CPSF6_HUMAN 500 HXC12_HUMAN 501 KDM4B_HUMAN 502 LMBL3_HUMAN 503 PHX2A_HUMAN 504 EMX1_HUMAN 505 NC2B_HUMAN 506 DLX4_HUMAN 507 SRY_HUMAN 508 ZN777_HUMAN 509 NELL1_HUMAN 510 ZN398_HUMAN 511 GATA3_HUMAN 512 BSH_HUMAN 513 SF3B4_HUMAN 514 TEAD1_HUMAN 515 TEAD3_HUMAN 516 RGAP1_HUMAN 517 PHF1_HUMAN 518 FOXA1_HUMAN 519 GATA2_HUMAN 520 FOXO3_HUMAN 521 ZN212_HUMAN 522 IRX4_HUMAN 523 ZBED6_HUMAN 524 LHX4_HUMAN 525 SIN3A_HUMAN 526 RBBP7_HUMAN 527 NKX61_HUMAN 528 TRI68_HUMAN 529 R51A1_HUMAN 530 MB3L1_HUMAN 531 DLX5_HUMAN 532 NOTC1_HUMAN 533 TERF2_HUMAN 534 ZN282_HUMAN 535 RGS12_HUMAN 536 ZN840_HUMAN 537 SPI2B_HUMAN_1 538 PAX7_HUMAN 539 NKX62_HUMAN 540 ASXL2_HUMAN 541 FOXO1_HUMAN 542 GATA3_HUMAN 543 GATA1_HUMAN 544 ZMYM5_HUMAN 545 ZN783_HUMAN 546 SPI2B_HUMAN_2 547 LRP1_HUMAN 548 MIXL1_HUMAN 549 SGT1_HUMAN 550 LMCD1_HUMAN 551 CEBPA_HUMAN 552 GATA2_HUMAN 553 SOX14_HUMAN 554 WTIP_HUMAN 555 PRP19_HUMAN 556 CBX6_HUMAN 557 NKX11_HUMAN 558 RBBP4_HUMAN 559 DMRT2_HUMAN 560 SMCA2_HUMAN 561 ZNF10_HUMAN 562 EED_HUMAN 563 RCOR1_HUMAN 564

A functional analog of any one of the above-listed proteins, i.e., a molecule having the same or substantially the same biological function (e.g., retaining 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more) of the protein's transcription factor function) is encompassed by the present disclosure. For example, the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and/or containing mutations relative to the above protein. In some embodiments, the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences listed in Table 4. Homologs, orthologs, and mutants of the above-listed proteins are also contemplated.

In certain embodiments, an epigenetic editor described herein comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627, and/or an effector domain derived from KAP1, MECP2, HP1a, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2, optionally wherein the parental protein is a human protein. In particular embodiments, an epigenetic editor described herein comprises a domain derived from KOX1, ZIM3, ZFP28, and/or ZN627, optionally wherein the parental protein is a human protein. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from KOX1 (ZNF10), e.g., a human KOX1. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZIM3 (ZNF657 or ZNF264), e.g., a human ZIM3. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZFP28, e.g., a human ZFP28. In certain embodiments, the epigenetic editor may comprise a KRAB domain derived from ZN627, e.g., a human ZN627. In certain embodiments, an epigenetic editor described herein may comprise a CDYL2, e.g., a human CDYL2, and/or a TOX domain (e.g., a human TOX domain) in combination with a KOX1 KRAB domain (e.g., a human KOX1 KRAB domain).

In certain embodiments, an epigenetic effector described herein comprises a repressor domain derived from KOX1/ZNF10 (SEQ ID NO: 89). For example, the repressor domain may comprise the sequence of SEQ ID NO: 89, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 89.

In certain embodiments, an epigenetic effector described herein comprises a repressor domain derived from KOX1/ZNF10, as shown in Table 5 below:

TABLE 5 Exemplary Effector Domains Derived from KOX1/ZNF10 Protein Protein Sequence KOX1/ZNF10 KRAB 1 SEQ ID NO: 565 KOX1/ZNF10 KRAB 2 SEQ ID NO: 566 KOX1/ZNF10 KRAB 3 SEQ ID NO: 567 KOX1/ZNF10 (aa 11-72) SEQ ID NO: 568 KOX1/ZNF10 (aa 11-108) SEQ ID NO: 569 KOX1/ZNF10 variant SEQ ID NO: 570 KOX1 KRAB-ZIM3 chimera SEQ ID NO: 571 ZIM3-KOX1 KRAB chimera SEQ ID NO: 572

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 565, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 565.

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 566, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 566.

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 567, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 567.

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 568, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 568.

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 569, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 569.

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 570, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 570.

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 571, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 571.

In particular embodiments, the repressor domain may comprise the amino acid sequence of SEQ ID NO: 572, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 572.

B. DNA Methyltransferases

In some embodiments, an effector domain of an epigenetic editor described herein alters target gene expression through DNA modification, such as methylation. Highly methylated areas of DNA tend to be less transcriptionally active than less methylated areas. DNA methylation occurs primarily at CpG sites (shorthand for “C-phosphate-G-” or “cytosine-phosphate-guanine” sites). Many mammalian genes have promoter regions near or including CpG islands (nucleic acid regions with a high frequency of CpG dinucleotides).

An effector domain described herein may be, e.g., a DNA methyltransferase (DNMT) or a catalytic domain thereof, or may be capable of recruiting a DNA methyltransferase. DNMTs encompass enzymes that catalyze the transfer of a methyl group to a DNA nucleotide, such as canonical cytosine-5 DNMTs that catalyze the addition of methyl groups to genomic DNA (e.g., DNMT1, DNMT3A, DNMT3B, and DNMT3C). This term also encompasses non-canonical family members that do not catalyze methylation themselves but that recruit (including activate) catalytically active DNMTs; a non-limiting examples of such a DNMT is DNMT3L. See, e.g., Lyko, Nat Review (2018) 19:81-92. Unless otherwise indicated, a DNMT domain may refer to a polypeptide domain derived from a catalytically active DNMT (e.g., DNMT1, DNMT3A, and DNMT3B) or from a catalytically inactive DNMT (e.g., DNMT3L). A DNMT may repress expression of the target gene through the recruitment of repressive regulatory proteins. In some embodiments, the methylation is at a CG (or CpG) dinucleotide sequence. In some embodiments, the methylation is at a CHG or CHH sequence, where H is any one of A, T, or C.

In some embodiments, a DNMT described herein can be an animal DNMT (e.g., a mammalian DNMT), a plant DNMT, a fungal DNMT, or a bacterial DNMT. A bacterial DNMT can be obtained from a bacterial species (e.g., a coccus bacterium, bacillus bacterium, spiral bacterium, or an intracellular, gram-positive, or gram-negative bacterium. In certain embodiments, the bacterial species is Mycoplasmatales bacterium, Mycoplasma marinum, or Spiroplasma chinense. In certain embodiments, the bacterial species is notM. penetrans, S. monbiae, H. parainfluenzae, A. luteus, H. aegyptius, H. haemolyticus, Moraxella, E. coli, T. aquaticus, C. crescentus, or C. difficile. In certain embodiments, an epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO: 601, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 601. In certain embodiments, an epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO: 602, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 602. In certain embodiments, an epigenetic editor described herein comprises a DNMT domain comprising SEQ ID NO: 603, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 603.

In certain embodiments, DNMTs in the epigenetic editors described herein may include, e.g., DNMT1, DNMT3A, DNMT3B, and/or DNMT3C. In some embodiments, the DNMT is a mammalian (e.g., human or murine) DNMT. In particular embodiments, the DNMT is DNMT3A (e.g., human DNMT3A). In certain embodiments, an epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 574, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 574. In certain embodiments, an epigenetic editor described herein comprises a DNMT3A domain comprising SEQ ID NO: 575, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 575. In some embodiments, the DNMT3A domain may have, e.g., a mutation at position H739 (such as H739A or H739E), R771 (such as R771L) and/or R836 (such as R836A or R836Q), or any combination thereof (numbering according to SEQ ID NO: 574).

In some embodiments, an effector domain described herein may be a DNMT-like domain. As used herein a “DNMT-like domain” is a regulatory factor of DNMT that may activate or recruit other DNMT domains, but does not itself possess methylation activity. In some embodiments, the DNMT-like domain is a mammalian (e.g., human or mouse) DNMT-like domain. In certain embodiments, the DNMT-like domain is DNMT3L, which may be, for example, human DNMT3L or mouse DNMT3L. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 578, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 578. In certain embodiments, an epigenetic editor herein comprises a DNMT3L domain comprising SEQ ID NO: 579, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 579. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 580, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 580. In certain embodiments, an epigenetic editor described herein comprises a DNMT3L domain comprising SEQ ID NO: 581, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 581. In some embodiments, the DNMT3L domain may have, e.g., a mutation corresponding to that at position D226 (such as D226V), Q268 (such as Q268K), or both (numbering according to SEQ ID NO: 578).

In certain embodiments, an epigenetic editor herein may comprise comprising both DNMT and DNMT-like effector domains. For example, the epigenetic editor may comprise a DNMT3A-3L domain, wherein DNMT3A and DNMT3L may be covalently linked. In other embodiments, an epigenetic editor described herein may comprise an effector domain that comprises only a DNMT3A domain (e.g., human DNMT3A), or only a DNMT-like domain (e.g., DNMT3L, which may be human or mouse DNMT3L).

Table 6 below provides exemplary DNMTs that may be part of an epigenetic effector domain described herein, or from which an effector domain of an epigenetic editor described herein may be derived.

TABLE 6 Exemplary DNMT Sequences Protein Name Species Target Protein Sequence DNMT1 Human 5mC SEQ ID NO: 573 DNMT3A (h3A) Human 5mC SEQ ID NO: 574 DNMT3A Human 5mC SEQ ID NO: 575 (catalytic domain) (h3As) DNMT3B Human 5mC SEQ ID NO: 576 DNMT3C Mouse 5mC SEQ ID NO: 577 DNMT3L (h3L) Human 5mC SEQ ID NO: 578 DNMT3L Human 5mC SEQ ID NO: 579 (catalytic domain) (h3Ls) DNMT3L (m3L) Mouse 5mC SEQ ID NO: 580 DNMT3L Mouse 5mC SEQ ID NO: 581 (catalytic domain) (m3Ls) DNMT3L Ailuropoda melanoleuca 5mC SEQ ID NO: 582 DNMT3L Ailuropoda melanoleuca 5mC SEQ ID NO: 583 (catalytic domain) DNMT3L Carlito syrichta 5mC SEQ ID NO: 584 DNMT3L Carlito syrichta 5mC SEQ ID NO: 585 (catalytic domain) DNMT3L Meriones unguiculatus 5mC SEQ ID NO: 586 DNMT3L Meriones unguiculatus 5mC SEQ ID NO: 587 (catalytic domain) DNMT3L Ochotona princeps 5mC SEQ ID NO: 588 DNMT3L Ochotona princeps 5mC SEQ ID NO: 589 (catalytic domain) DNMT3L Neosciurus carolinensis 5mC SEQ ID NO: 590 DNMT3L Neosciurus carolinensis 5mC SEQ ID NO: 591 (catalytic domain) DNMT3L Bison bison 5mC SEQ ID NO: 592 DNMT3L Bison bison 5mC SEQ ID NO: 593 (catalytic domain) DNMT3L Equus przewalskii 5mC SEQ ID NO: 594 DNMT3L Equus przewalskii 5mC SEQ ID NO: 595 (catalytic domain) DNMT3L Mus caroli 5mC SEQ ID NO: 596 DNMT3L Mus caroli 5mC SEQ ID NO: 597 (catalytic domain) DNMT3L Pan troglodytes 5mC SEQ ID NO: 598 DNMT3L Pan troglodytes 5mC SEQ ID NO: 599 (catalytic domain) TRDMT1 Human tRNA 5mC SEQ ID NO: 600 (DNMT2) DNA cytosine Mycoplasmatales 5mC SEQ ID NO: 601 methyltransferase bacterium DNA cytosine Mycoplasma marinum 5mC SEQ ID NO: 602 methyltransferase DNA (cytosine-5-)- Spiroplasma chinense 5mC SEQ ID NO: 603 methyltransferase M.MpeI Mycoplasma penetrans 5mC SEQ ID NO: 604 M.SssI Spiroplasma monobiae 5mC SEQ ID NO: 605 M.HpaII Haemophilus 5mC (CCGG) SEQ ID NO: 606 parainfluenzae M.AluI Arthrobacter luteus 5mC (AGCT) SEQ ID NO: 607 M.HaeIII Haemophilus aegyptius 5mC (GGCC) SEQ ID NO: 608 M.HhaI Haemophilus 5mC (GCGC) SEQ ID NO: 609 haemolyticus M.MspI Moraxella 5mC (CCGG) SEQ ID NO: 610 Masc1 Ascobolus 5mC SEQ ID NO: 611 MET1 Arabidopsis 5mC SEQ ID NO: 612 Masc2 Ascobolus 5mC SEQ ID NO: 613 Dim-2 Neurospora 5mC SEQ ID NO: 614 dDnmt2 Drosophila 5mC SEQ ID NO: 615 Pmt1 S. pombe 5mC SEQ ID NO: 616 DRM1 Arabidopsis 5mC SEQ ID NO: 617 DRM2 Arabidopsis 5mC SEQ ID NO: 618 CMT1 Arabidopsis 5mC SEQ ID NO: 619 CMT2 Arabidopsis 5mC SEQ ID NO: 620 CMT3 Arabidopsis 5mC SEQ ID NO: 621 Rid Neurospora 5mC SEQ ID NO: 622 hsdM gene bacteria (E. coli, strain 12) m6A SEQ ID NO: 623 hsdS gene bacteria (E. coli, strain 12) m6A SEQ ID NO: 624 M.TaqI Bacteria (Thermus m6A SEQ ID NO: 625 aquaticus) M.EcoDam E. coli m6A SEQ ID NO: 626 M.CcrMI Caulobacter crescentus m6A SEQ ID NO: 627 CamA Clostridioides difficile m6A SEQ ID NO: 628

A functional analog of any one of the above-listed proteins, i.e., a molecule having the same or substantially the same biological function (e.g., retaining 700% or more, 8000 or more, 9000 or more, 9500 or more, or 98% or more) of the protein's DNA methylation function or recruiting function) is encompassed by the present disclosure. For example, the functional analog may be an isoform or a variant of the above-listed protein, e.g., containing a portion of the above protein with or without additional amino acid residues and/or containing mutations relative to the above protein. In some embodiments, the functional analog has a sequence identity that is at least 75, 80, 85, 90, 95, 98, or 99% to one of the sequences listed in Table 6. In some embodiments, the effector domain herein comprises only the functional domain (or functional analog thereof), e.g., the catalytic domain or recruiting domain, of an above-listed protein. In some embodiments, the effector domain herein comprises one or more epigenetic effector domains selected from Table 6, or functional homologs, orthologs, or variants thereof.

As used herein, a DNMT domain (e.g., a DNMT3A domain or a DNMT3L domain) refers to a protein domain that is identical to the parental protein (e.g., a human or murine DNMT3A or DNMT3L) or a functional analog thereof (e.g., having a functional fragment, such as a catalytic fragment or recruiting fragment, of the parental protein; and/or having mutations that improve the activity of the DNMT protein).

An epigenetic editor herein may effect methylation at, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more CpG dinucleotide sequences in the target gene or chromosome. The CpG dinucleotide sequences may be located within or near the target gene in CpG islands, or may be located in a region that is not a CpG island. A CpG island generally refers to a nucleic acid sequence or chromosome region that comprises a high frequency of CpG dinucleotides. For example, a CpG island may comprise at least 50% GC content. The CpG island may have a high observed-to-expected CpG ratio, for example, an observed-to-expected CpG ratio of at least 60%. As used herein, an observed-to-expected CpG ratio is determined by Number of CpG*(sequence length)/(Number of C*Number of G). In some embodiments, the CpG island has an observed-to-expected CpG ratio of at least 60%, 70%, 80%, 90% or more. A CpG island may be a sequence or region of, e.g., at least 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 nucleotides. In some embodiments, only 1, or less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 CpG dinucleotides are methylated by the epigenetic editor.

In some embodiments, an epigenetic editor herein effects methylation at a hypomethylated nucleic acid sequence, i.e., a sequence that may lack methyl groups on the 5-methyl cytosine nucleotides (e.g., in CpG) as compared to a standard control. Hypomethylation may occur, for example, in aging cells or in cancer (e.g., early stages of neoplasia) relative to a younger cell or non-cancer cell, respectively.

In some embodiments, an epigenetic editor described herein induces methylation at a hypermethylated nucleic acid sequence.

In some embodiments, methylation may be introduced by the epigenetic editor at a site other than a CpG dinucleotide. For example, the target gene sequence may be methylated at the C nucleotide of CpA, CpT, or CpC sequences. In some embodiments, an epigenetic editor comprises a DNMT3A domain and effects methylation at CpG, CpA, CpT, CpC sequences, or any combination thereof. In some embodiments, an epigenetic editor comprises a DNMT3A domain that lacks a regulatory subdomain and only maintains a catalytic domain. In some embodiments, the epigenetic editor comprising a DNMT3A catalytic domain effects methylation exclusively at CpG sequences. In some embodiments, an epigenetic editor comprising a DNMT3A domain that comprises a mutation, e.g. a R836A or R836Q mutation (numbering according to SEQ ID NO: 574), has higher methylation activity at CpA, CpC, and/or CpT sequences as compared to an epigenetic editor comprising a wildtype DNMT3A domain.

C. Histone Modifiers

In some embodiments, an effector domain of an epigenetic editor herein mediates histone modification. Histone modifications play a structural and biochemical role in gene transcription, such as by formation or disruption of the nucleosome structure that binds to the histone and prevents gene transcription. Histone modifications may include, for example, acetylation, deacetylation, methylation, phosphorylation, ubiquitination, SUMOylation and the like, e.g., at their N-terminal ends (“histone tails”). These modifications maintain or specifically convert chromatin structure, thereby controlling responses such as gene expression, DNA replication, DNA repair, and the like, which occur on chromosomal DNA. Post-translational modification of histones is an epigenetic regulatory mechanism and is considered essential for the genetic regulation of eukaryotic cells. Recent studies have revealed that chromatin remodeling factors such as SWI/SNF, RSC, NURF, NRD, and the like, which facilitate transcription factor access to DNA by modifying the nucleosome structure; histone acetyltransferases (HATs) that regulate the acetylation state of histones; and histone deacetylases (HDACs), act as important regulators.

In particular, the unstructured N-termini of histones may be modified by acetylation, deacetylation, methylation, ubiquitylation, phosphorylation, SUMOylation, ribosylation, citrullination O-GlcNAcylation, crotonylation, or any combination thereof. For example, histone acetyltransferases (HATs) utilize acetyl-CoA as a cofactor and catalyze the transfer of an acetyl group to the epsilon amino group of the lysine side chains. This neutralizes the lysine's positive charge and weakens the interactions between histones and DNA, thus opening the chromosomes for transcription factors to bind and initiate transcription. Acetylation of K14 and K9 lysines of histone H3 by histone acetyltransferase enzymes may be linked to transcriptional competence in humans. Lysine acetylation may directly or indirectly create binding sites for chromatin-modifying enzymes that regulate transcriptional activation. On the other hand, histone methylation of lysine 9 of histone H3 may be associated with heterochromatin, or transcriptionally silent chromatin.

In certain embodiments, an effector domain of an epigenetic editor described herein comprises a histone methyltransferase domain. The effector domain may comprise, for example, a DOT1L domain, a SET domain, a SUV39H1 domain, a G9a/EHMT2 protein domain, an EZH1 domain, an EZH2 domain, a SETDB1 domain, or any combination thereof. In particular embodiments, the effector domain comprises a histone-lysine-N-methyltransferase SETDB1 domain.

In some embodiments, the effector domain comprises a histone deacetylase protein domain. In certain embodiments, the effector domain comprises a HDAC family protein domain, for example, a HDAC1, HDAC3, HDAC5, HDAC7, or HDAC9 protein domain. In particular embodiments, the effector domain comprises a nucleosome remodeling and deacetylase complex (NURD), which removes acetyl groups from histones.

D. Other Effector Domains

In some embodiments, the effector domain comprises a tripartite motif containing protein (TRIM28, TIF1-beta, or KAP1). In certain embodiments, the effector domain comprises one or more KAP1 proteins. A KAP1 protein in an epigenetic editor herein may form a complex with one or more other effector domains of the epigenetic editor or one or more proteins involved in modulation of gene expression in a cellular environment. For example, KAP1 may be recruited by a KRAB domain of a transcriptional repressor. A KAP1 protein domain may interact with or recruit one or more protein complexes that reduces or silences gene expression. In some embodiments, KAP1 interacts with or recruits a histone deacetylase protein, a histone-lysine methyltransferase protein, a chromatin remodeling protein, and/or a heterochromatin protein. For example, a KAP1 protein domain may interact with or recruit a heterochromatin protein 1 (HP1) protein, a SETDB1 protein, an HDAC protein, and/or a NuRD protein complex component. In some embodiments, a KAP1 protein domain interacts with or recruits a ZFP90 protein (e.g., isoform 2 of ZFP90), and/or a FOXP3 protein. An exemplary KAP1 amino acid sequence is shown in SEQ ID NO: 629.

In some embodiments, the effector domain comprises a protein domain that interacts with or is recruited by one or more DNA epigenetic marks. For example, the effector domain may comprise a methyl CpG binding protein 2 (MECP2) protein that interacts with methylated DNA nucleotides in the target gene (which may or may not be at a CpG island of the target gene). An MECP2 protein domain in an epigenetic editor described herein may induce condensed chromatin structure, thereby reducing or silencing expression of the target gene. In some embodiments, an MECP2 protein domain in an epigenetic editor described herein may interact with a histone deacetylase (e.g., HDAC), thereby repressing or silencing expression of the target gene. In some embodiments, an MECP2 protein domain in an epigenetic editor described herein may block access of a transcription factor or transcriptional activator to the target sequence, thereby repressing or silencing expression of the target gene. An exemplary MECP2 amino acid sequence is shown in SEQ ID NO: 630.

Also contemplated as effector domains for the epigenetic editors described herein are, e.g., a chromoshadow domain, a ubiquitin-2 like Rad60 SUMO-like (Rad60 -SLD/SUMO) domain, a chromatin organization modifier domain (Chromo) domain, a Yaf2/RYBP C-terminal binding motif domain (YAF2_RYBP), a CBX family C-terminal motif domain (CBX7_C), a zinc finger C3HC4 type (RING finger) domain (ZF-C3HC4_2), a cytochrome b5 domain (Cyt-b5), a helix-loop-helix domain (HLH), a helix-hairpin-helix motif domain (e.g., HHH_3), a high mobility group box domain (HMG-box), a basic leucine zipper domain (e.g., bZIP_1 or bZIP 2), a Myb_DNA-binding domain, a homeodomain, a MYM-type zinc finger with FCS sequence domain (ZF-FCS), an interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), an SSX repressor domain (SSXRD), a B-box-type zinc finger domain (ZF-B box), a CXXC zinc finger domain (ZF-CXXC), a regulator of chromosome condensation 1 domain (RCC1), an SRC homology 3 domain (SH3_9), a sterile alpha motif domain (SAM 1), a sterile alpha motif domain (SAM 2), a sterile alpha motif/Pointed domain (SAM_PNT), a Vestigial/Tondu family domain (Vg_Tdu), a LIM domain, an RNA recognition motif domain (RRM_1), a paired amphipathic helix domain (PAH), a proteasomal ATPase OB C-terminal domain (Prot_ATP_ID_OB), a nervy homology 2 domain (NHR2), a hinge domain of cleavage stimulation factor subunit 2 (CSTF2_hinge), a PPAR gamma N-terminal region domain (PPARgamma N), a CDC48 N-terminal domain (CDC48_2), a WD40 repeat domain (WD40), a FipI motif domain (FipI), a PDZ domain (PDZ_6), a Von Willebrand factor type C domain (VWC), a NAB conserved region 1 domain (NCD1), an Si RNA-binding domain (S1), an HNF3 C-terminal domain (HNF_C), a Tudor domain (Tudor 2), a histone-like transcription factor (CBF/NF-Y) and archaeal histone domain (CBFD_NFYB_HMF), a zinc finger protein domain (DUF3669), an EGF-like domain (cEGF), a GATA zinc finger domain (GATA), a TEA/ATTS domain (TEA), a phorbol esters/diacylglycerol binding domain (C1-1), polycomb-like MTF2 factor 2 domain (Mtf2_C), a transactivation domain of FOXO protein family (FOXO-TAD), a homeobox KN domain (Homeobox_KN), a BED zinc finger domain (ZF-BED), a zinc finger of C3HC4-type RING domain (ZF-C3HC4_4), a RAD51 interacting motif domain (RAD51_interact), a p55-binding region of a methyl-CpG-binding domain protein MBD (MBDa), a Notch domain, a Raf-like Ras-binding domain (RBD), a Spin/Ssty family domain (Spin-Ssty), a PHD finger domain (PHD_3), a Low-density lipoprotein receptor domain class A (Ldl_recept_a), a CS domain, a DM DNA-binding domain, and a QLQ domain.

In some embodiments, the effector domain is a protein domain comprising a YAF2_RYBP domain or homeodomain or any combination thereof. In certain embodiments, the homeodomain of the YAF2_RYBP domain is a PRD domain, an NKL domain, a HOXL domain, or a LIM domain. In particular embodiments, the YAF2_RYBP domain may comprise a 32 amino acid Yaf2/RYBP C-terminal binding motif domain (32 aa RYBP).

In some embodiments, the effector domain comprises a protein domain selected from a group consisting of SUMO3 domain, Chromo domain from M phase phosphoprotein 8 (MPP8), chromoshadow domain from Chromobox 1 (CBX1), and SAM_1/SPM domain from Scm Polycomb Group Protein Homolog 1 (SCMH1).

In some embodiments, the effector domain comprises an HNF3 C-terminal domain (HNF_C). The HNF_C domain may be from FOXA1 or FOXA2. In certain embodiments, the HNF_C domain comprises an EH1 (engrailed homology 1) motif.

In some embodiments, the effector domain may comprise an interferon regulatory factor 2-binding protein zinc finger domain (IRF-2BP1_2), a Cyt-b5 domain from DNA repair factor HERC2 E3 ligase, a variant SH3 domain (SH39) from Bridging Integrator 1 (BIN1), an HMG-box domain from transcription factor TOX or ZF-C3HC4_2 RING finger domain from the polycomb component PCGF2, a Chromodomain-helicase-DNA binding protein 3 (CHD3) domain, or a ZNF783 domain.

IV. Epigenetic Editors

Provided herein are epigenetic editors (i.e., epigenetic editing systems) that direct epigenetic modification(s) to a target sequence in a gene of interest, e.g., using one or more DNA-binding domains as described herein and one or more effector domains (e.g., epigenetic repressor domains) as described herein, in any combination. The DNA-binding domain (in concert with a guide polynucleotide such as one described herein, where the DNA-binding domain is a polynucleotide guided DNA-binding domain) directs the effector domain to epigenetically modify the target sequence, resulting in gene repression or silencing that may be durable and inheritable across cell generations. In some aspects, the epigenetic editors described herein can repress or silence genes reversibly or irreversibly in cells.

In particular embodiments, an epigenetic editor described herein comprises one or more fusion proteins, each comprising (1) DNA-binding domain(s) and (2) effector domain(s). The effector domains may be on one or more fusion proteins comprised by the epigenetic editor. For example, a single fusion protein may comprise all of the effector domains with a DNA-binding domain. Alternatively, the effector domains or subsets thereof may be on separate fusion proteins, each with a DNA-binding domain (which may be the same or different). A fusion protein described herein may further comprise one or more linkers (e.g., peptide linkers), detectable tags, nuclear localization signals (NLSs), or any combination thereof. As used herein, a “fusion protein” refers to a chimeric protein in which two or more coding sequences (e.g., for DNA-binding domain(s) and/or effector domain(s)) are covalently or non-covalently joined, directly or indirectly.

In some embodiments, an epigenetic editor described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more effector (e.g., repression/repressor) domains, which may be identical or different. In certain embodiments, two or more of said effector domains function synergistically. Combinations of effector domains may comprise DNA methylation domains, histone deacetylation domains, histone methylation domains, and/or scaffold domains that recruit any of the above. For example, an epigenetic editor described herein may comprise one or more transcriptional repressor domains (e.g., a KRAB domain such as KOX1, ZIM3, ZFP28, or ZN627 KRAB) in combination with one or more DNA methylation domains (e.g., a DNMT domain) and/or recruiter domain (e.g., a DNMT3L domain). Such an epigenetic editor may comprise, for instance, a KRAB domain, a DNMT3A domain, and a DNMT3L domain. In some embodiments, the epigenetic editor further comprises an additional effector domain (e.g., a KAP1, MECP2, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, RBBP4, RCOR1, or SCML2 domain). In some embodiments, the additional effector domain is a CDYL2, TOX, TOX3, TOX4, or HP1a domain. For example, an epigenetic editor described herein may comprise a CDYL2 and/or a TOX domain in combination with a KRAB domain (e.g., a KOX1 KRAB domain).

A. Linkers

A fusion protein as described herein may comprise one or more linkers that connect components of the epigenetic editor. A linker may be a peptide or non-peptide linker.

In some embodiments, one or more linkers utilized in an epigenetic editor provided herein is a peptide linker, i.e., a linker comprising a peptide moiety. A peptide linker can be any length applicable to the epigenetic editor fusion proteins described herein. In some embodiments, the linker can comprise a peptide between 1 and 200 (e.g., between 1 and 80) amino acids. In some embodiments, the linker comprises from 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 1 to 80, 1 to 100, 1 to 150, 1 to 200, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 60, 5 to 80, 5 to 100, 5 to 150, 5 to 200, 10 to 20, 10 to 30, 10 to 40, 10 to 50, to 60, 10 to 80, 10 to 100, 10 to 150, 10 to 200, 20 to 30, 20 to 40, 20 to 50, 20 to 60, 20 to 80, 20 to 100, 20 to 150, 20 to 200, 30 to 40, 30 to 50, 30 to 60, 30 to 80, 30 to 100, 30 to 150, 30 to 200, 40 to 50, 40 to 60, 40 to 80, 40 to 100, 40 to 150, 40 to 200, 50 to 60 50 to 80, 50 to 100, 50 to 150, 50 to 200, 60 to 80, 60 to 100, 60 to 150, 60 to 200, 80 to 100, 80 to 150, 80 to 200, 100 to 150, 100 to 200, or 150 to 200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the peptide linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. For example, the peptide linker may be 4, 5, 16, 20, 24, 27, 32, 40, 64, 92, or 104 amino acids in length. The peptide linker may be a flexible or rigid linker. In particular embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 631-637 and 664-665 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

In certain embodiments, the peptide linker is an XTEN linker. Such a linker may comprise part of the XTEN sequence (Schellenberger et al., Nat Biotechnol (2009) 27(1):1186-90), an unstructured hydrophilic polypeptide consisting only of residues G, S, P, T, E, and A. The term “XTEN” as used herein refers to a recombinant peptide or polypeptide lacking hydrophobic amino acid residues. XTEN linkers typically are unstructured and comprise a limited set of natural amino acids. Fusion of XTEN to proteins alters its hydrodynamic properties and reduces the rate of clearance and degradation of the fusion protein. These XTEN fusion proteins are produced using recombinant technology, without the need for chemical modifications, and degraded by natural pathways. The XTEN linker may be, for example, 5, 10, 16, 20, 26, or 80 amino acids in length. In some embodiments, the XTEN linker is 16 amino acids in length. In some embodiments, the XTEN linker is 80 amino acids in length. In certain embodiments, the XTEN linker may be XTEN10, XTEN16, XTEN20, or XTEN80. In certain embodiments, the XTEN linker may comprise the amino acid sequence of any one of SEQ ID NOs: 638-643 or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In particular embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 638. In particular embodiments, the XTEN linker comprises the amino acid sequence of SEQ ID NO: 643.

In some embodiments, one or more linkers utilized in an epigenetic editor provided herein is a non-peptide linker. For example, the linker may be a carbon bond, a disulfide bond, or carbon-heteroatom bond. In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, or branched or unbranched aliphatic or heteroaliphatic linker.

In some embodiments, one or more linkers utilized in an epigenetic editor provided herein is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). The linker may comprise, for example, a monomer, dimer, or polymer of aminoalkanoic acid; an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.); a monomer, dimer, or polymer of aminohexanoic acid (Ahx); or a polyethylene glycol moiety (PEG); or an aryl or heteroaryl moiety. In certain embodiments, the linker may be based on a carbocyclic moiety (e.g., cyclopentane or cyclohexane) or a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

Various linker lengths and flexibilities can be employed between any two components of an epigenetic editor (e.g., between an effector domain (e.g., a repressor domain) and a DNA-binding domain (e.g., a Cas9 domain), between a first effector domain and a second effector domain, etc.). The linkers may range from very flexible linkers, such as glycine/serine-rich linkers, to more rigid linkers, in order to achieve the optimal length for effector domain activity for the specific application. In some embodiments, the more flexible linkers are glycine/serine-rich linkers (GS-rich linkers), where more than 45% (e.g., more than 48, 50, 55, 60, 70, 80, or 90%) of the residues are glycine or serine residues. Non-limiting examples of the GS-rich linkers are (GGGGS)n (SEQ ID NO: 664), (G)n, and W linker (SEQ ID NO: 637). In some embodiments, the more rigid linkers are in the form of the form (EAAAK)n (SEQ ID NO: 665), (SGGS)n (SEQ ID NO: 631, and (XP)n). In the aforementioned formulae of flexible and rigid linkers, n may be any integer between 1 and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (GGGGS)n motif, wherein n is 4 (SEQ ID NO: 636).

In some embodiments, a linker in an epigenetic editor described herein comprises a nuclear localization signal, for example, with the amino acid sequence of any one of SEQ ID NOs: 644-649. In some embodiments, a linker in an epigenetic editor described herein comprises an expression tag, e.g., a detectable tag such as a green fluorescent protein.

B. Nuclear Localization Signals

A fusion protein described herein may comprise one or more nuclear localization signals, and in certain embodiments, may comprise two or more nuclear localization signals. For example, the fusion protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nuclear localization signals. As used herein, a “nuclear localization signal” (NLS) is an amino acid sequence that directs proteins to the nucleus. In certain embodiments, the NLS may be an SV40 NLS (e.g., with the amino acid sequence of SEQ ID NO: 644). The fusion protein may comprise an NLS at its N-terminus, C-terminus, or both, and/or an NLS may be embedded in the middle of the fusion protein (e.g., at the N- or C-terminus of a DNA-binding domain or an effector domain).

In some embodiments, the fusion protein may comprise two NLSs. The fusion protein may comprise two NLSs at its N-terminus or C-terminus. The fusion protein may comprise one NLS located at its N-terminus and one NLS embedded in the middle of the fusion protein, or one NLS located at its C-terminus and one NLS embedded in the middle of the fusion protein. The fusion protein may comprise two NLSs embedded in the middle of the fusion protein.

In some embodiments, the fusion protein may comprise four NLSs. The fusion protein may comprise at least two (e.g., two, three, or four) NLSs at its N-terminus or C-terminus. The fusion protein may comprise at least one (e.g., one, two, three, or four) NLSs embedded in the middle of the fusion protein. In particular embodiments, the fusion protein may comprise two NLSs at its N-terminus and two NLSs at its C-terminus.

An NLS described herein may be an endogenous NLS sequence. In certain embodiments, an NLS described herein comprises the amino acid sequence of any one of SEQ ID NOs: 644-649, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the selected sequence. In particular embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 644. Additional NLSs are known in the art.

In some embodiments, an epigenetic editor comprising a fusion protein that comprises at least one NLS at the N-terminus and at least one NLS at the C-terminus may increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more as compared to an epigenetic editor with a corresponding fusion protein that does not have at least one NLS at the N-terminus and at least one NLS at the C-terminus.

In some embodiments, an epigenetic editor comprising a fusion protein that comprises two NLSs at the N-terminus and two NLSs at the C-terminus may increase the efficiency of the epigenetic editor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1,000%, at least 5,000%, at least 10,000%, at least 50,000%, at least 100,000%, or more as compared to an epigenetic editor with a corresponding fusion protein that does not have two NLSs at the N-terminus and two NLSs at the C-terminus.

C. Tags

Epigenetic editors provided herein may comprise one or more additional sequences (“tags”) for tracking, detection, and localization of the editors. In some embodiments, the epigenetic editor comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more detectable tags. Each of the detectable tags may be the same or different.

For example, an epigenetic editor fusion protein may comprise cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, poly-histidine tags (also referred to as histidine tags or His-tags), maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1 or Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art.

D. Fusion Protein Configurations

A fusion protein of an epigenetic editor described herein may have its components structured in different configurations. For example, the DNA-binding domain may be at the C-terminus, the N-terminus, or in between two or more epigenetic effector domains or additional domains. In some embodiments, the DNA-binding domain is at the C-terminus of the epigenetic editor. In some embodiments, the DNA-binding domain is at the N-terminus of the epigenetic editor. In some embodiments, the DNA-binding domain is linked to one or more nuclear localization signals. In some embodiments, the DNA-binding domain is flanked by an epigenetic effector domain and/or an additional domain on both sides. In some embodiments, where “DBD” indicates DNA-binding domain and “ED” indicates effector domain, the epigenetic editor comprises the configuration of:

In some embodiments, an epigenetic editor comprises a DNA-binding domain (DBD), a DNA methyltransferase (DNMT) domain, and a transcriptional repressor (“repressor”) domain that represses or silences expression of a target gene. The DBD, DNMT, and transcriptional repressor domains may be any as described herein, in any combination. The DBD, DNMT domain, and repressor domain may be in any configuration, e.g., with any of said domains at the N-terminus, at the C-terminus, or in the middle of the fusion protein. In some embodiments, the epigenetic editor comprises a fusion protein with the configuration of:

In some embodiments, a connecting structure”]-[“in any one of the epigenetic editor structures is a linker, e.g., a peptide linker; a detectable tag; a peptide bond; a nuclear localization signal; and/or a promoter or regulatory sequence. In an epigenetic editor structure, the multiple connecting structures “]-[” may be the same or may each be a different linker, tag, NLS, or peptide bond. In some embodiments, the DNMT domain may comprise any one of the domains in Table 6, or any combinations or homologs thereof. In particular embodiments, the DNMT domain comprises DNMT3A or a truncated version thereof, DNMT3L or a truncated version thereof, or both. In particular embodiments, the DBD is a catalytically inactive polynucleotide guided DNA-binding domain (e.g., a dCas9) or a ZFP domain. In certain embodiments, the repressor domain comprises any one of the domains shown in Table 4 or 5, or any combinations or homologs thereof. For example, the repressor domain may be a KRAB domain. In certain embodiments, the repressor domain is a ZFP28, ZN627, KAP1, MeCP2, HP1b, CBX8, CDYL2, TOX, Tox3, Tox4, EED, RBBP4, RCOR1, or SCML2 domain, or a fusion of two of said domains (e.g., a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB). In particular embodiments, the repressor domain is a KRAB domain from ZFP28, ZN627, ZIM3, or KOX1.

In some embodiments, the epigenetic editor comprises a configuration selected from

wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure]-[is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and/or a regulatory sequence. The DBD, repressor, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. For example, the DNMT3A and DNMT3L domains may be selected from those in Table 6. In particular embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain; the repressor domain is a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.

In some embodiments, the epigenetic editor comprises a configuration selected from

wherein [DNMT3A-DNMT3L] indicates that the DNMT3A and DNMT3L domains are directly fused via a peptide bond, and wherein the connecting structure]-[is any one of the linkers as described herein, a detectable tag, an affinity domain, a peptide bond, a nuclear localization signal, a promoter, and/or a regulatory sequence. The DBD, SETDB1, DNMT3A, and DNMT3L domains may be any as described herein, in any combination. In particular embodiments, the DBD is a CRISPR-associated protein domain (e.g., dCas9) or a ZFP domain; the SETDB1 domain is derived from human SETDB1, ZIM3, ZFP28, or ZN627; the DNMT3A domain is a human DNMT3A domain; and the DNMT3L domain is a human or mouse DNMT3L domain; any combination of these components is also contemplated by the present disclosure.

Particular constructs contemplated herein include:

The DNMT3L and DNMT3A may be derived from human parental proteins, mouse parental proteins, or any combination thereof. In certain embodiments, the DNMT3L and DNMT3A are derived from mouse and human parental proteins, respectively (mDNMT3L and hDNMT3A). In certain embodiments, the DNMT3L and DNMT3A are both derived from human parental proteins (hDNMT3L and hDNMT3A). In some embodiments, the dCas9 is dSpCas9. In some embodiments, the KOX1 is human KOX1. Also contemplated is any of Configurations 1-6 wherein the KOX1 KRAB domain is replaced by a ZFP28, ZN627, or ZIM3 KRAB domain. In some embodiments, the ZFP28, ZN627, and ZIM3 are human ZFP28, ZN627, and ZIM3, respectively. In particular embodiments, the fusion construct may have the configuration:

In particular embodiments, a fusion construct described herein may have Configuration 1 and comprise SEQ ID NO: 658, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 658 below, the XTEN linkers are underlined, the W linker is bolded, underlined, and italicized, the NLS sequences are bolded, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the dCas9 domain is bolded and italicized, and the KOX1 KRAB domain is underlined and bolded:

(SEQ ID NO: 658) MNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGI QVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGP FDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKE GDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHR ARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITT RSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNM SRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPS FSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVL KSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQ PLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLT EDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAP LTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEPKKKRK VYMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSN EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYP TIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIE RMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV EDRENASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQN EKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSID NKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKY DENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAY LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKAT AKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITI MERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM LASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQSITGLYETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTG RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY QLTKPDVILRLEKGEEP

In particular embodiments, a fusion construct described herein may comprise the sequence provided below (SEQ ID NO: 1495), or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 1495 below, the XTEN linkers are underlined, the W linker is bolded, underlined, and italicized, the NLS sequences are bolded, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the dCas9 domain is bolded and italicized, and the KOX1 KRAB domain is underlined and bolded:

(SEQ ID NO: 1495) MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGI QVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGP FDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKE GDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHR ARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITT RSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNM SRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPS FSSGLVPLSLRGSHMNPLEMFETVPVWRRQPVRVLSLFEDIKKEL TSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPEDLVYGATPPL GHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKE DLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALV SEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELT SSLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEPKKKRKV YMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER MTNEDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF LSGEQKKAIVDLLEKTNRKVTVKQLKEDYFKKIECFDSVEISGVE DRENASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNE KLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYD ENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFV EQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE QAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTGR TLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQ LTKPDVILRLEKGEEP

In particular embodiments, a fusion construct described herein may have Configuration 2 and comprise SEQ ID NO: 659, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 659 below, the XTEN linkers are underlined, the W linker is bolded, underlined, and italicized, the NLS sequences are bolded and underlined, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the ZFP domain is bolded, and the KOX1 KRAB domain is underlined and bolded. Variable amino acids represented by Xs are the amino acids of the DNA-recognition helix of the zinc finger and XX in italics may be either TR, LR or LK.

(SEQ ID NO: 659) MNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGI QVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGP FDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKE GDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHR ARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITT RSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNM SRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPS FSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVL KSLGFLESGSGSGGGTLKYVEDVINVVRRDVEKWGPFDLVYGSTQ PLGSSCDRCPGWYMFQFHRILQYALPRQESQRPFFWIFMDNLLLT EDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAP LTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEPKKKRK VYSRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMR NFSXXXXXXXHXXTH[linker]PFQCRICMRNFSXXXXXXXHXX THTGEKPFQCRICMRNFSXXXXXXXHXXTH[linker]PFQCRIC MRNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTH LRGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTRE EWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEE P

In certain embodiments, the six “XXXXXXX” regions in SEQ ID NO: 659 comprise, in order, the F1-F6 amino acid sequences shown in Table 1 for any one of ZF001-ZF048. [linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 651). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 652). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 651 and the other linker sequence may have the amino acid sequence of SEQ ID NO: 652.

In particular embodiments, a fusion construct described herein may comprise the sequence provided below (SEQ ID NO: 1496), or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In SEQ ID NO: 1496 below, the XTEN linkers are underlined, the W linker is bolded, underlined, and italicized, the NLS sequences are bolded and underlined, the DNMT3A sequence is italicized, the DNMT3L sequence is underlined and italicized, the ZFP domain is bolded, and the KOX1 KRAB domain is underlined and bolded. Variable amino acids represented by Xs are the amino acids of the DNA-recognition helix of the zinc finger and XX in italics may be either TR, LR or LK.

(SEQ ID NO: 1496) MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGI QVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGP FDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKE GDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHR ARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITT RSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNM SRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPS FSSGLVPLSLRGSHMNPLEMFETVPVWRRQPVRVLSLFEDIKKEL TSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPL GHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKE DLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALV SEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELT SSLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEPKKKRKV YSRPGERPFQCRICMRNFSXXXXXXXHXXTHTGEKPFQCRICMRN FSXXXXXXXHXXTH[linker]PFQCRICMRNFSXXXXXXXHXXT HTGEKPFQCRICMRNFSXXXXXXXHXXTH[linker]PFQCRICM RNFSXXXXXXXHXXTHTGEKPFQCRICMRNFSXXXXXXXHXXTHL RGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREE WKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP

In certain embodiments, the six “XXXXXXX” regions in SEQ ID NO: 1496 comprise, in order, the F1-F6 amino acid sequences shown in Table 1 for any one of ZF001-ZF048. [linker] represents a linker sequence. In some embodiments, one or both linker sequences may be TGSQKP (SEQ ID NO: 651). In some embodiments, one or both linker sequences may be TGGGGSQKP (SEQ ID NO: 652). In some embodiments, one linker sequence may have the amino acid sequence of SEQ ID NO: 651 and the other linker sequence may have the amino acid sequence of SEQ ID NO: 652.

In some embodiments, the fusion protein may further comprise a Dnmt3A ADD domain, e.g., downstream of the Dnmt3A domain sequence disclosed in SEQ ID Nos 658, 659, 1495, or 1496 disclosed above. In some embodiments, the ADD sequence is situated between the Dnmt3A and the Dnmt3L sequence of the fusion protein. In some embodiments, the ADD sequence is at the C-terminal end of the Dnmt3A domain. In some embodiments, the Dnmt3A sequence and the ADD sequence are separated by a linker, e.g., a linker disclosed herein. In some embodiments, the ADD sequence and the Dnmt3L sequence are separated by a linker, e.g., a linker disclosed herein. In some embodiments, the ADD domain comprises the sequence:

(SEQ ID NO: 1497) MAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKAN QRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLYDDD GYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVH AMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFE TVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVT DTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYA RPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGG SLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWP TKLVKNCFLPLREYFKYFSTELTSSL.

In particular embodiments, a fusion construct described herein may have Configuration 7 and comprise SEQ ID NO: 660, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

In particular embodiments, a fusion construct described herein may have Configuration 9 and comprise SEQ ID NO: 661, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

In particular embodiments, a fusion construct described herein may have Configuration 11 and comprise SEQ ID NO: 662, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

In particular embodiments, a fusion construct described herein may have Configuration 13 and comprise SEQ ID NO: 663, or a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

In some embodiments, a fusion construct described herein (e.g., the fusion construct of any one of Configurations 1-14) is within an expression construct that comprises a WPRE sequence, a polyadenylation site, or both. In certain embodiments, the WPRE sequence is in a 3′ noncoding region. In certain embodiments, the WPRE sequence is upstream from a poly-adenylation site. In particular embodiments, the expression construct comprises the fusion construct (e.g., of any one of Configurations 1-14) and a WPRE sequence in a 3′ noncoding region upstream from a polyadenylation site.

In some embodiments, a fusion construct described herein may have the sequence of any one of Fusion Proteins 1-12 as shown in Example 12.

Multiple fusion proteins may be used to effect activation or repression of a target gene or multiple target genes. For example, an epigenetic editor fusion protein comprising a DNA-binding domain (e.g., a dCas9 domain) and an effector domain may be co-delivered with two or more guide polynucleotides (e.g., gRNAs), each targeting a different target DNA sequence. The target sites for two of the DNA-binding domains may be the same or in the vicinity of each other, or separated by, for example, about 100 base pairs, about 200 base pairs, about 300 base pairs, about 400 base pairs, about 500 base pairs, or about 600 or more base pairs. In addition, when targeting double-strand DNA, such as an endogenous gene locus, the guide polynucleotides may target the same or different strands (one or more to the positive strand and/or one or more to the negative strand).

V. Target Sequences

An epigenetic editor herein may be directed to a target sequence in PCSK9 to effect epigenetic modification of the PCSK9 gene. As used herein, a “target sequence,” a “target site,” or a “target region” is a nucleic acid sequence present in a gene of interest; in some instances, the target sequence may be outside but in the vicinity of the gene of interest wherein methylation or binding by a repressor of the target sequence represses expression of the gene. In some embodiments, the target sequence may be a hypomethylated or hypermethylated nucleic acid sequence.

The target sequence may be in any part of a target gene. In some embodiments, the target sequence is part of or near a noncoding sequence of the gene. In some embodiments, the target sequence is part of an exon of the gene. In some embodiments, the target sequence is part of or near a transcriptional regulatory sequence of the gene, such as a promoter or an enhancer. In some embodiments, the target sequence is adjacent to, overlaps with, or encompasses a CpG island. In certain embodiments, the target sequence is within about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 base pairs (bp) flanking a PCSK9 TSS. In certain embodiments, the target sequence is within 500 bp flanking the PCSK9 TSS. In certain embodiments, the target sequence is within 1000 bp flanking the PCSK9 TSS.

In some embodiments, the target sequence may hybridize to a guide polynucleotide sequence (e.g., gRNA) complexed with a fusion protein comprising a polynucleotide guided DNA-binding domain (e.g., a CRISPR protein such as dCas9) and effector domain(s). The guide polynucleotide sequence may be designed to have complementarity to the target sequence, or identity to the opposing strand of the target sequence. In some embodiments, the guide polynucleotide comprises a spacer sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a protospacer sequence in the target sequence. In particular embodiments, the guide polynucleotide comprises a spacer sequence that is 100% identical to a protospacer sequence in the target sequence.

In some embodiments, where the DNA-binding domain of an epigenetic editor described herein is a zinc finger array, the target sequence may be recognized by said zinc finger array.

In some embodiments, where the DNA-binding domain of an epigenetic editor described herein is a TALE, the target sequence may be recognized by said TALE.

A target sequence described herein may be specific to one copy of a target gene, or may be specific to one allele of a target gene. Accordingly, the epigenetic modification and modulation of expression thereof may be specific to one copy or one allele of the target gene. For example, an epigenetic editor may repress expression of a specific copy harboring a target sequence recognized by the DNA-binding domain (e.g., a copy associated with a disease or condition, or that harbors a mutation associated with a disease or condition).

In some embodiments, the target PCSK9 genomic region may fall within the sequence shown below (chr1:55038548-55040548), with or without the terminal A:

(SEQ ID NO: 1488) TACCTCATGGAGTCACTGTCAACCCACTGGTTGCACTGTCTTTGT GCACTGGCTCTCTGGAGTGAGGTCTTTGCAAACAAAGTGGAAAGA GCATCAACTTTGGACTCCAGCACCTAGATTCAGAGCAGGCCATTT CACTCGGAATCTGCTGTGCATCTGCAAGGGAGGATCATAAATTCG CCTTTGTTTCTTCCCAGTATCGACAGCCCTTCCAGAAAGAGCAAG CCTCATGTCATGCCACATGTACAATCTGAGGCCAGGAGCTCTCTT TCCCCTTTTCATCCTCCTGCCTGGTACACAATAGGTGTTTACTGG ATGCTTGTCCAGTTGATTTCTTGAACATGGTGTGTAAAAGGAATC TTTGCAAATTGAATCTTCTGGAAAGCTGAGCTTGTGCCTACCATA GAATTCTGAATGTACCTATATGACGTCTTTGCAAACTTAAAACCT GAATCTTTGTAGTATAAATCCCTTGAAATGCATGTAGGCTGGACA TCAAAAGCAAGCAATCTCTTCAAGGAGCAGCTAGTTGGTAAGGTC AGTGTGCAGGGTGCATAAAGGGCAGAGGCCGGAGGGGGTCCAGGC TAAGTTTAGAAGGCTGCCAGGTTAAGGCCAGTGGAAAGAATTCGG TGGGCAGCGAGGAGTCCACAGTAGGATTGATTCAGAAGTCTCACT GGTCAGCAGGAGACAAGGTGGACCCAGGAAACACTGAAAAGGTGG GCCCGGCAGAACTTGGAGTCTGGCATCCCACGCAGGGTGAGAGGC GGGAGAGGAGGAGCCCCTAGGGCGCCGGCCTGCCTTCCAGCCCAG TTAGGATTTGGGAGTTTTTTCTTCCCTCTGCGCGTAATCTGACGC TGTTTGGGGAGGGCGAGGCCGAAACCTGATCCTCCAGTCCGGGGG TTCCGTTAATGTTTAATCAGATAGGATCGTCCGATGGGGCTCTGG TGGCGTGATCTGCGCGCCCCAGGCGTCAAGCACCCACACCCTAGA AGGTTTCCGCAGCGACGTCGAGGCGCTCATGGTTGCAGGCGGGCG CCGCCGTTCAGTTCAGGGTCTGAGCCTGGAGGAGTGAGCCAGGCA GTGAGACTGGCTCGGGCGGGCCGGGACGCGTCGTTGCAGCAGCGG CTCCCAGCTCCCAGCCAGGATTCCGCGCGCCCCTTCACGCGCCCT GCTCCTGAACTTCAGCTCCTGCACAGTCCTCCCCACCGCAAGGCT CAAGGCGCCGCCGGCGTGGACCGCGCACGGCCTCTAGGTCTCCTC GCCAGGACAGCAACCTCTCCCCTGGCCCTCATGGGCACCGTCAGC TCCAGGCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTG CTGCTCCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGAC GGCGACTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGAC GGCCTGGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCAC CGCTGCGCCAAGGTGCGGGTGTAGGGATGGGAGGCCGGGGCGAAC CCGCAGCCGGGACGGTGCGGTGCTGTTTCCTCTCGGGCCTCAGTT TCCCCCCATGTAAGAGAGGAAGTGGAGTGCAGGTCGCCGAGGGCT CTTCGCTTGGCACGATCTTGGGGACTGCAGGCAAGGCGGCGGGGG AGGACGGGTAGTGGGGAGCACGGTGGAGAGCGGGGACGGCCGGCT CTTTGGGGACTTGCTGGGGCGTGCGGCTGCGCTATTCAGTGGGAA GGTTCGCGGGGTTGGGAGACCCGGAGGCCGAGGAAGGGCGAGCAG AGCACTGCCAGGATATCCTGCCCAGATTTCCCAGTTTCTGCCTCG CCGCGGCACAGGTGGGTGAAGGAGTGAATGCCTGGAACGTACTGG GAACTGCACCAGGCACAGAGAAAGCGGGCTTGCCATTATAGTGGG TTCCGATTTGGTTTGGAAAACATGGGCAGCGGAGGGTGGAGGGCC TGGAGAGAAGGCCCTACCCGA

In some embodiments, the target sequence may be GRCh38 Chr1:55039228-55040296, as shown below:

(SEQ ID NO: 1489) GCAGGAGACAAGGTGGACCCAGGAAACACTGAAAAGGTGGGCCCG GCAGAACTTGGAGTCTGGCATCCCACGCAGGGTGAGAGGCGGGAG AGGAGGAGCCCCTAGGGCGCCGGCCTGCCTTCCAGCCCAGTTAGG ATTTGGGAGTTTTTTCTTCCCTCTGCGCGTAATCTGACGCTGTTT GGGGAGGGCGAGGCCGAAACCTGATCCTCCAGTCCGGGGGTTCCG TTAATGTTTAATCAGATAGGATCGTCCGATGGGGCTCTGGTGGCG TGATCTGCGCGCCCCAGGCGTCAAGCACCCACACCCTAGAAGGTT TCCGCAGCGACGTCGAGGCGCTCATGGTTGCAGGCGGGCGCCGCC GTTCAGTTCAGGGTCTGAGCCTGGAGGAGTGAGCCAGGCAGTGAG ACTGGCTCGGGCGGGCCGGGACGCGTCGTTGCAGCAGCGGCTCCC AGCTCCCAGCCAGGATTCCGCGCGCCCCTTCACGCGCCCTGCTCC TGAACTTCAGCTCCTGCACAGTCCTCCCCACCGCAAGGCTCAAGG CGCCGCCGGCGTGGACCGCGCACGGCCTCTAGGTCTCCTCGCCAG GACAGCAACCTCTCCCCTGGCCCTCATGGGCACCGTCAGCTCCAG GCGGTCCTGGTGGCCGCTGCCACTGCTGCTGCTGCTGCTGCTGCT CCTGGGTCCCGCGGGCGCCCGTGCGCAGGAGGACGAGGACGGCGA CTACGAGGAGCTGGTGCTAGCCTTGCGTTCCGAGGAGGACGGCCT GGCCGAAGCACCCGAGCACGGAACCACAGCCACCTTCCACCGCTG CGCCAAGGTGCGGGTGTAGGGATGGGAGGCCGGGGCGAACCCGCA GCCGGGACGGTGCGGTGCTGTTTCCTCTCGGGCCTCAGTTTCCCC CCATGTAAGAGAGGAAGTGGAGTGCAGGTCGCCGAGGGCTCTTCG CTTGGCACGATCTTGGGGACTGCAGGCAAGGCGGCGGGGGAGGAC GGGTAGTGGGGAGCACGGTGGAGAGCGGGGACGGCCGGCTCTTTG GGGACTTGCTGGGGCGTGCGGCTGCGCTATTCAG

VI. Epigenetic Modifications

An epigenetic editor described herein may perform sequence-specific epigenetic modification(s) (e.g., alteration of chemical modification(s)) of a target gene that harbors the target sequence. Such epigenetic modulation may be safer and more easily reversible than modulation due to gene editing, e.g., with generation of DNA double-strand breaks. In some embodiments, the epigenetic modulation may reduce or silence the target gene. In some embodiments, the modification is at a specific site of the target sequence. In some embodiments, the modification is at a specific allele of the target gene. Accordingly, the epigenetic modification may result in modulated (e.g., reduced) expression of one copy of a target gene harboring a specific allele, and not the other copy of the target gene. In some embodiments, the specific allele is associated with a disease, condition, or disorder.

In some embodiments, the epigenetic modification reduces or abolishes transcription of the target gene harboring the target sequence. In some embodiments, the epigenetic modification reduces or abolishes transcription of a copy of the target gene harboring a specific allele recognized by the epigenetic editor. In some embodiments, the epigenetic editor reduces the level of or eliminates expression of a protein encoded by the target gene. In some embodiments, the epigenetic editor reduces the level of or eliminates expression of a protein encoded by a copy of the target gene harboring a specific allele recognized by the epigenetic editor. The target PCSK9 gene may be epigenetically modified in vitro, ex vivo, or in vivo.

The effector domain of an epigenetic editor described herein may alter (e.g., deposit or remove) a chemical modification at a nucleotide of the target gene or at a histone associated with the target gene. The chemical modification may be altered at a single nucleotide or a single histone, or may be altered at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or more nucleotides.

In some embodiments, an effector domain of an epigenetic editor described herein may alter a CpG dinucleotide within the target gene. In some embodiments, all CpG dinucleotides within 2000, 1500, 1000, 500, or 200 bps flanking a target sequence (e.g., in an alteration site as described herein) are altered according to a modification type described herein, as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more of the CpG dinucleotides are altered as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the CpG dinucleotides are altered as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor. In some embodiments, one single CpG dinucleotide is altered, as compared to the original state of the gene or the gene in a comparable cell not contacted with the epigenetic editor.

An effector domain of an epigenetic editor described herein may alter a histone modification state of a histone associated with or bound to the target gene. For example, an effector domain may deposit a modification on one or more lysine residues of histone tails of histones associated with the target gene. In some embodiments, the effector domain may result in deacetylation of one or more histone tails of histones associated with the target gene, thereby reducing or silencing expression of the target gene. In some embodiments, the histone modification state is a methylation state. For example, the effector domain may result in a H3K9, H3K27 or H4K20 methylation (e.g. one or more of a H3K9me2, H3K9me3, H3K27me2, H3K27me3, and H4K20me3 methylation) at one or more histone tails associated with the target gene, thereby reducing or silencing expression of the target gene.

In some embodiments, all histone tails of histones bound to DNA nucleotides within 2000, 1500, 1000, 500, or 200 bps flanking the target sequence are altered according to a modification type as described herein, as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of histone tails of the bound histones are altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. For example, one single histone tail of the bound histones may be altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor. As another example, one single bound histone octamer may be altered as compared to the original state of the chromosome or the chromosome in a comparable cell not contacted with the epigenetic editor.

The chemical modification deposited at target gene DNA nucleotides or histone residues may be at or in close proximity to a target sequence in the target gene. In some embodiments, an effector domain of an epigenetic editor described herein alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide 100-200, 200-300, 300-400, 400-55, 500-600, 600-700, or 700-800 nucleotides 5′ or 3′ to the target sequence in the target gene. In some embodiments, an effector domain alters a chemical modification state of a nucleotide or histone tail bound to a nucleotide within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides flanking the target sequence. As used herein, “flanking” refers to nucleotide positions 5′ to the 5′ end of and 3′ to the 3′ end of a particular sequence, e.g. a target sequence.

In some embodiments, an effector domain mediates or induces a chemical modification change of a nucleotide or a histone tail bound to a nucleotide distant from a target sequence. Such modification may be initiated near the target sequence, and may subsequently spread to one or more nucleotides in the target gene distant from the target sequence. For example, an effector domain may initiate alteration of a chemical modification state of one or more nucleotides or one or more histone residues bound to one or more nucleotides within 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nucleotides flanking the target sequence, and the chemical modification state alteration may spread to one or more nucleotides at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or more nucleotides from the target sequence in the target gene, either upstream or downstream of the target sequence. In certain embodiments, the chemical modification may be initiated at less than 2, 3, 5, 10, 20, 30, 40, 50, or 100 nucleotides in the target gene and spread to at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or more nucleotides in the target gene. In some embodiments, the chemical modification spreads to nucleotides in the entire target gene. Additional proteins or transcription factors, for example, transcription repressors, methyltransferases, or transcription regulation scaffold proteins, may be involved in the spreading of the chemical modification. Alternatively, the epigenetic editor alone may be involved.

In some embodiments, an epigenetic editor described herein reduces expression of a target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the target gene in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject (e.g., in the absence of the epigenetic editor). In some embodiments, the epigenetic editors described herein reduces expression of a copy of target gene by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more, as measured by transcription of the copy of the target gene in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject. In certain embodiments, the copy of the target gene harbors a specific sequence or allele recognized by the epigenetic editor. In particular embodiments, the epigenetically modified copy encodes a functional protein, and accordingly an epigenetic editor disclosed herein may reduce or abolish expression and/or function of the protein. For example, an epigenetic editor described herein may reduce expression and/or function of a protein encoded by the target gene by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100 fold in a cell, a tissue, or a subject as compared to a control cell, control tissue, or a control subject.

Modulation of target gene expression can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels; changes in protein activity; changes in product levels; changes in downstream gene expression; changes in transcription or activity of reporter genes such as, for example, luciferase, CAT, beta-galactosidase, or GFP; changes in signal transduction; changes in phosphorylation and dephosphorylation; changes in receptor-ligand interactions; changes in concentrations of second messengers such as, for example, cGMP, cAMP, IP3, and Ca²⁺; changes in cell growth; changes in neovascularization; and/or changes in any functional effect of gene expression. Measurements can be made in vitro, in vivo, and/or ex vivo, and can be made by conventional methods, e.g., measurement of RNA or protein levels, measurement of RNA stability, and/or identification of downstream or reporter gene expression. Readout can be by way of, for example, chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3), changes in intracellular calcium levels; cytokine release, and the like.

Methods for determining the expression level of a gene, for example the target of an epigenetic editor, may include, e.g., determining the transcript level of a gene by reverse transcription PCR, quantitative RT-PCR, droplet digital PCR (ddPCR), Northern blot, RNA sequencing, DNA sequencing (e.g., sequencing of complementary deoxyribonucleic acid (cDNA) obtained from RNA); next generation (Next-Gen) sequencing, nanopore sequencing, pyrosequencing, or Nanostring sequencing. Levels of protein expressed from a gene may be determined, e.g., by Western blotting, enzyme linked immuno-absorbance assays, mass-spectrometry, immunohistochemistry, or flow cytometry analysis. Gene expression product levels may be normalized to an internal standard such as total messenger ribonucleic acid (mRNA) or the expression level of a particular gene, e.g., a housekeeping gene.

In some embodiments, the effect of an epigenetic editor in modulating target gene expression may be examined using a reporter system. For example, an epigenetic editor may be designed to target a reporter gene encoding a reporter protein, such as a fluorescent protein. Expression of the reporter gene in such a model system may be monitored by, e.g., flow cytometry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy. In some embodiments, a population of cells may be transfected with a vector that harbors a reporter gene. The vector may be constructed such that the reporter gene is expressed when the vector transfects a cell. Suitable reporter genes include genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins. The population of cells carrying the reporter system may be transfected with DNA, mRNA, or vectors encoding the epigenetic editor targeting the reporter gene.

VII. Pharmaceutical Compositions

In one aspect, the present disclosure provides a pharmaceutical composition comprising as an active ingredient (or as the sole active ingredient) one or more epigenetic editors described herein or component(s) (e.g., fusion proteins and/or guide polynucleotides) thereof, or nucleic acid molecule(s) encoding said epigenetic editors or component(s) thereof. For example, a pharmaceutical composition may comprise nucleic acid molecule(s) encoding the fusion protein(s) (and guide polynucleotides, where applicable) of an epigenetic editor described herein. In some embodiments, separate pharmaceutical compositions comprise the fusion protein(s) and the guide polynucleotide(s). A pharmaceutical composition may also comprise cells that have undergone epigenetic modification(s) mediated or induced by an epigenetic editor provided herein.

Generally, the epigenetic editors described herein or component(s) thereof, or nucleic acid molecule(s) encoding said epigenetic editors or component(s) thereof, of the present disclosure are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable excipient(s), e.g., as described below.

The term “excipient” is used herein to describe any ingredient other than the compound(s) of the present disclosure. The choice of excipient(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.

Formulations of a pharmaceutical composition suitable for parenteral administration typically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.

VIII. Delivery Methods

In some embodiments, the epigenetic editor or its component(s) are introduced to target cells in the form of nucleic acid molecule(s) encoding the epigenetic editor or its component(s); accordingly, the pharmaceutical compositions herein comprise the nucleic acid molecule(s). Such nucleic acid molecule(s) may be, for example, DNA, RNA or mRNA, and/or modified nucleic acid sequence(s) (e.g., with chemical modifications, a 5′ cap, or one or more 3′ modifications). In some embodiments, the nucleic acid molecule(s) may be delivered as naked DNA or RNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by target cells. In some embodiments, the nucleic acid molecule(s) may be in nucleic acid expression vector(s), which may include expression control sequences such as promoters, enhancers, transcription signal sequences, transcription termination sequences, introns, polyadenylation signals, Kozak consensus sequences, internal ribosome entry sites (IRES), etc. Such expression control sequences are well known in the art. A vector may also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein.

Examples of vectors include, but are not limited to, plasmid vectors; viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, or spleen necrosis virus, vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and other recombinant vectors. In certain embodiments, the vector is a plasmid or a viral vector. Viral particles or virus-like particles (VLPs) may also be used to deliver nucleic acid molecule(s) encoding epigenetic editors or component(s) thereof as described herein. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles may also be engineered to incorporate targeting ligands to alter target tissue specificity.

In certain embodiments, an epigenetic editor as described herein or component(s) thereof are encoded by nucleic acid sequence(s) present in one or more viral vectors, or a suitable capsid protein of any viral vector. Examples of viral vectors include adeno-associated viral vectors (e.g., derived from AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAV10, and/or variants thereof); retroviral vectors (e.g., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD100), lentiviral vectors (e.g., HIV and FIV-based vectors), and herpesvirus vectors (e.g., HSV-2).

In some embodiments, delivery involves an adeno-associated virus (AAV) vector. AAV vector delivery may be particularly useful where the DNA-binding domain of an epigenetic editor fusion protein is a zinc finger array. Without wishing to be bound by any theory, the smaller size of zinc finger arrays compared to larger DNA-binding domains such as Cas protein domains may allow such a fusion protein to be conveniently packed in viral vectors such as an AAV vector.

Any AAV serotype, e.g., human AAV serotype, can be used for an AAV vector as described herein, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), and AAV serotype 11 (AAV 11), as well as variants thereof. In some embodiments, an AAV variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a wildtype AAV. In certain embodiments, the AAV variant may be engineered such that its capsid proteins have reduced immunogenicity or enhanced transduction ability in humans. In some instances, one or more regions of at least two different AAV serotype viruses are shuffled and reassembled to generate a chimeric variant. For example, a chimeric AAV may comprise inverted terminal repeats (ITRs) that are of a heterologous serotype compared to the serotype of the capsid. The resulting chimeric AAV can have a different antigenic reactivity or recognition compared to its parental serotypes. In some embodiments, a chimeric variant of an AAV includes amino acid sequences from 2, 3, 4, 5, or more different AAV serotypes.

Non-viral systems are also contemplated for delivery as described herein. Non-viral systems include, but are not limited to, nucleic acid transfection methods including electroporation, sonoporation, calcium phosphate transfection, microinjection, DNA biolistics, lipid-mediated transfection, transfection through heat shock, compacted DNA-mediated transfection, lipofection, cationic agent-mediated transfection, and transfection with liposomes, immunoliposomes, exosomes, or cationic facial amphiphiles (CFAs). In certain embodiments, one or more mRNAs encoding epigenetic editor fusion proteins as described herein may be co-electroporated with one or more guide polynucleotides (e.g., gRNAs) as described herein. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic (e.g., lipid) or inorganic (e.g., gold). For instance, organic (e.g. lipid and/or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure.

In some embodiments, delivery is accomplished using a lipid nanoparticle (LNP). LNP compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. In some embodiments, a LNP refers to any particle that has a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. In some embodiments, a nanoparticle may range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.

An LNP as described herein may be made from cationic, anionic, or neutral lipids. In some embodiments, an LNP may comprise neutral lipids, such as the fusogenic phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or the membrane component cholesterol, as helper lipids to enhance transfection activity and nanoparticle stability. In some embodiments, an LNP may comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. The lipids may be combined in any molar ratios to produce the LNP. In some embodiments, the LNP is a liver-targeting (e.g., preferentially or specifically targeting the liver) LNP.

Any type of cell may be targeted for delivery of an epigenetic editor or component(s) thereof as described herein. For example, the cells may be eukaryotic or prokaryotic. In some embodiments, the cells are mammalian (e.g., human) cells. Human cells may include, for example, hepatocytes, biliary epithelial cells (cholangiocytes), stellate cells, Kupffer cells, and liver sinusoidal endothelial cells.

In some embodiments, an epigenetic editor described herein, or component(s) thereof, are delivered to a host cell for transient expression, e.g., via a transient expression vector. Transient expression of the epigenetic editor or its component(s) may result in prolonged or permanent epigenetic modification of the target gene. For example, the epigenetic modification may be stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, or 12 weeks or more; or 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, after introduction of the epigenetic editor into the host cell. The epigenetic modification may be maintained after one or more mitotic and/or meiotic events of the host cell. In particular embodiments, the epigenetic modification is maintained across generations in offspring generated or derived from the host cell.

IX. Therapeutic Uses of Epigenetic Editors

The present disclosure also provides methods for treating or preventing a condition in a subject, comprising administering to the subject an epigenetic editor or pharmaceutical composition as described herein. The epigenetic editor may effect an epigenetic modification of a target polynucleotide sequence in a target gene associated with a disease, condition, or disorder in the subject, thereby modulating expression of the target gene to treat or prevent the disease, condition, or disorder. In some embodiments, the epigenetic editor reduces the expression of the target gene to an extent sufficient to achieve a desired effect, e.g., a therapeutically relevant effect such as the prevention or treatment of the disease, condition, or disorder.

In some embodiments, a subject is administered a system for modulating (e.g., repressing) expression of PCSK9, wherein the system comprises (1) the fusion protein(s) and, where relevant, guide polynucleotide(s) of an epigenetic editor as described herein, or (2) nucleic acid molecules encoding said fusion protein(s) and, where relevant, guide polynucleotide(s).

“Treat”, “treating” and “treatment” refer to a method of alleviating or abrogating a biological disorder and/or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and/or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment. In some embodiments, as compared with an equivalent untreated control, alleviating a symptom may involve reduction of the symptom by at least 3%, 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 100% as measured by any standard technique.

In some embodiments, the subject may be a mammal, e.g., a human. In some embodiments, the subject is selected from a non-human primate such as chimpanzee, cynomolgus monkey, or macaque, and other ape and monkey species.

In some embodiments, the human patient has a condition selected from hypercholesterolemia (e.g., familial hypercholesterolemia such as heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), or established atherosclerotic cardiovascular disease (ASCVD)) or renal insufficiency (RI).

In some embodiments, a patient to be treated with an epigenetic editor of the present disclosure has received prior treatment for the condition to be treated (e.g., hypercholesterolemia (such as HeFH, HoFH, HF, or established ASCVD) or RI). In other embodiments, the patient has not received such prior treatment. In some embodiments, the patient has failed on a prior treatment for the condition (e.g., a prior hypercholesterolemia treatment).

An epigenetic editor of the present disclosure may be administered in a therapeutically effective amount to a patient with a condition described herein. “Therapeutically effective amount,” as used herein, refers to an amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated, and/or result in clinical endpoint(s) desired by healthcare professionals. An effective amount for therapy may be measured by its ability to stabilize disease progression and/or ameliorate symptoms in a patient, and preferably to reverse disease progression. The ability of an epigenetic editor of the present disclosure to reduce or silence PCSK9 expression may be evaluated by in vitro assays, e.g., as described herein, as well as in suitable animal models that are predictive of the efficacy in humans. Suitable dosage regimens will be selected in order to provide an optimum therapeutic response in each particular situation, for example, administered as a single bolus or as a continuous infusion, and with possible adjustment of the dosage as indicated by the exigencies of each case.

An epigenetic editor of the present disclosure may be administered without additional therapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with an epigenetic editor of the present disclosure may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the additional therapeutic agent is any known in the art to treat hypercholesterolemia or RI. Therapeutic agents include, but are not limited to, statins, fibrates, HMG-CoA reductase inhibitors, niacin, bile acid modulators or sequestrants, cholesterol absorption inhibitors or modulators, CETP inhibitors, MTTP inhibitors, and PPAR agonists.

The epigenetic editors or components thereof (or nucleic acid molecules encoding the epigenetic editors or components thereof) of the present disclosure may be administered by any method accepted in the art, e.g., subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously, intralymphatically, or intraperitoneally. In particular embodiments, a pharmaceutical composition of the present disclosure is administered intravenously to the subject.

X. Definitions

The term “nucleic acid” as used herein refers to any oligonucleotide or polynucleotide containing nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double-strand form, and includes DNA and RNA. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group, and are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which include natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modified versions which place new reactive groups such as amines, alcohols, thiols, carboxylates, alkylhalides, etc. Nucleic acids may contain known nucleotide analogs and/or modified backbone residues or linkages, which may be synthetic, naturally occurring, and non-naturally occurring. Such nucleotide analogs, modified residues, and modified linkages are well known in the art, and may provide a nucleic acid molecule with enhanced cellular uptake, reduced immunogenicity, and/or increased stability in the presence of nucleases.

As used herein, an “isolated” or “purified” nucleic acid molecule is a nucleic acid molecule that exists apart from its native environment. For example, an “isolated” or “purified” nucleic acid molecule (1) has been separated away from the nucleic acids of the genomic DNA or cellular RNA of its source of origin; and/or (2) does not occur in nature. In some embodiments, an “isolated” or “purified” nucleic acid molecule is a recombinant nucleic acid molecule.

It will be understood that in addition to the specific proteins and nucleic acid molecules mentioned herein, the present disclosure also contemplates the use of variants, derivatives, homologs, and fragments thereof. A variant of any given sequence may have the specific sequence of residues (whether amino acid or nucleic acid residues) modified in such a manner that the polypeptide or polynucleotide in question substantially retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally-occurring sequence (in some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues). For specific proteins described herein (e.g., KRAB, dCas9, DNMT3A, and DNMT3L proteins described herein), the present disclosure also contemplates any of the protein's naturally occurring forms, or variants or homologs that retain at least one of its endogenous functions (e.g., at least 50%, 60%, 70%, 80%, 90%, 85%, 96%, 97%, 98%, or 99% of its function as compared to the specific protein described).

Some exemplary fusion proteins embraced by the present disclosure are provided herein. It will be appreciated by the skilled artisan, that these exemplary proteins are non-limiting examples and that additional proteins are within the scope of the present disclosure. For example, where fusion exemplary proteins comprising a specific domain, e.g., a mammalian DNMT3A, DNMT3L and/or KRAB domain, such as a human or mouse DNMT3A, DNMT3L and/or KRAB domain, are provided, the skilled artisan will be able to ascertain that, in some embodiments, fusion proteins with the same configuration, but with one or more of the mammalian domains substituted for a homologous domain from another mammal, e.g., one or more mouse domains substituted for one or more human domains, are also embraced by the present disclosure. For example, where an exemplary fusion protein is provided that comprises a mouse DNMT3L domain, a fusion protein of the same architecture but with the mouse DNMT3L substituted for a human DNMT3L domain is also embraced.

As used herein, a homologue of any polypeptide or nucleic acid sequence contemplated herein includes sequences having a certain homology with the wildtype amino acid and nucleic sequence. A homologous sequence may include a sequence, e.g. an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85%, 90%, 91%, 92%<93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the subject sequence. The term “percent identical” in the context of amino acid or nucleotide sequences refers to the percent of residues in two sequences that are the same when aligned for maximum correspondence. In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%, or 100%) of the reference sequence. Sequence identity may be measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

The percent identity of two nucleotide or polypeptide sequences is determined by, e.g., BLAST® using default parameters (available at the U.S. National Library of Medicine's National Center for Biotechnology Information website). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90%) of the reference sequence.

It will be understood that the numbering of the specific positions or residues in polypeptide sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues.

The term “modulate” or “alter” refers to a change in the quantity, degree, or extent of a function. For example, an epigenetic editor as described herein may modulate the activity of a promoter sequence by binding to a motif within the promoter, thereby inducing, enhancing, or suppressing transcription of a gene operatively linked to the promoter sequence. As other examples, an epigenetic editor as described herein may block RNA polymerase from transcribing a gene, or may inhibit translation of an mRNA transcript. The terms “inhibit,” “repress,” “suppress,” “silence” and the like, when used in reference to an epigenetic editor or a component thereof as described herein, refers to decreasing or preventing the activity (e.g., transcription) of a nucleic acid sequence (e.g., a target gene) or protein relative to the activity of the nucleic acid sequence or protein in the absence of the epigenetic editor or component thereof. The term may include partially or totally blocking activity, or preventing or delaying activity. The inhibited activity may be, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% less than that of a control, or may be, e.g., at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold less than that of a control.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” should be assumed to mean an acceptable error range for the particular value.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise indicated, the recitation of a listing of elements herein includes any of the elements singly or in any combination. The recitation of an embodiment herein includes that embodiment as a single embodiment, or in combination with any other embodiment(s) herein. All publications, patents, patent applications, and other references mentioned herein are incorporated by reference in their entirety. To the extent that references incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed compositions and methods in any manner.

Some protein sequences, e.g., some fusion protein sequences, provided herein include a peptide tag, e.g., a His6 tag, or a DYKDDDDK (SEQ ID NO: 1528) tag, which are useful for detection and/or purification of tagged proteins, but do not affect protein function. These peptide tags, and additional suitable peptide tags, are well known to those of skill in the art. It will be apparent to the person of skill in the art that the disclosed tags can be substituted for other suitable peptide tags, and that fusion proteins of the same or highly similar sequence, but not including such peptide tags, e.g., from which the peptide tag has been cleaved or which are created without a peptide tag, are suitable for carrying out embodiments of the present disclosure as well.

In order that the present disclosure may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present disclosure in any manner.

EXAMPLES Example 1: Fusion Protein Design and Synthesis

Fusion proteins comprising dCas9, DNMT3A, DNMT3L, and KOX1 KRAB (“CRISPR-of”) were designed and constructed. From N terminus to C terminus, the proteins have the domains DNMT3A-linker-DNMT3L-XTEN80-NLS-dSpCas9-NLS-XTEN16-KOX1 KRAB (SEQ ID NOs: 658 and 1495). The CRISPR-off plasmid construct has been described in Nunez (Nunez et al., Cell (2021) 184(9):2503-19) and was ordered from Twist Biosciences.

ZF fusion proteins (“ZF-off”) comprising DNMT3A, 3L, and KOX1 KRAB were also constructed. The constructs have the general structure DNMT3A-linker-DNMT3L-XTEN80-NLS-ZFP domain-NLS-XTEN16-KOX1Krab (SEQ ID NOs: 659 and 1496).

Example 2: Selection of Target PCSK9 Sequences for gRNA Epigenetic Silencing

gRNAs targeting+/−1 kb from the PCSK9 TSS were computationally designed using the Benchling gRNA platform (Benchling (2021), retrieved from benchling.com) for human (GRCh38), mouse (mm10) and Macacafascicularis (5.0) PCSK9. gRNAs containing poly-TTTT sequences were first discarded. We performed gRNA off-target analysis using CasOFFinder (Bae et al., Bioinformatics (2014) 30(10):1473-5). gRNAs were discarded if they matched to multiple locations across the respective genome build for each independent species.

A cross-reactivity sequence analysis was performed on human PCSK9 gRNAs in order to annotate sequence mismatches with Macaca or mouse gRNA sequences. In particular, gRNA sequence alignments were performed to identify the degree of DNA similarity at each nucleotide, including the annotation of guides that contain up to zero, one, or two nucleotide mismatches. A final set of 226 gRNA sequences was selected for the PCSK9 primary screen in HeLa cells.

Example 3: Selection of ZF Target Sites and Design of ZF Proteins for Epigenetic Silencing

A library of two-finger ZFPs (2F units), each recognizing 6 bp DNA sites, was used to design larger six-finger ZFP arrays targeting 18 bp DNA binding sites. The source of the 2F units was a set of three-finger zinc finger proteins that had been selected to bind specific target sites using a bacterial-2-hybrid (B2H) selection system (Hurt et al., PNAS (2003) 100:12271-6; Maeder et al., Mol Cell (2008) 31(2):294-301). A list of targetable DNA sites was created by generating all possible triplet combinations of 6 bp binding sites represented in the library and allowing either 0 or 1 bp between the 6 bp target sites. To identify zinc finger target sites within PCSK9, the sequence+/−1 kb from TSS (human (GRCh38)) was interrogated against this list. For each identified ZF target site, multiple ZF proteins could be designed. Design of the six recognition helices used to generate the full proteins was performed by selecting two-finger units and taking into account a number of factors such as known binding preferences of zinc finger proteins, the frequency with which amino acids in positions −1, 2, 3 and 6 had been selected in the B2H selection system to bind the desired target base, avoidance of amino acids in positions −1, 2, 3 and 6 that had been selected to bind multiple different bases in the B2H, and maintaining context dependencies by matching flanking bases where possible. The full ZF sequence is derived from the naturally occurring Zif268 protein and selected recognition helices were maintained in the sequence context in which they were selected in the B2H (either fingers 1-2 or fingers 2-3 from Zif268). Two-finger units were joined by the linker TGSQKP (SEQ ID NO: 651) where 6 bp binding sites were contiguous and by the linker TGGGGSQKP (SEQ ID NO: 652) where 1 bp separated the 6 bp binding sites. A final set of 209 ZFPs targeted to 49 distinct binding sites were selected for the PCSK9 primary screen in HeLa cells.

FIG. 1 shows the overlap of the gRNAs and zinc finger proteins mapped to the PCSK9 target region.

Example 4: Guide RNA Screening in HeLa Cells

A primary screen of gRNAs targeting PCSK9 was performed in HeLa cells. gRNA sequences were ordered from Twist Biosciences as DNA fragments with a u6 promoter sequence preceding the gRNA coding sequence.

HeLa cells were transfected with gRNA and CRISPR-off in DNA format. Six 96-well plates (Sigma-Aldrich Catalog No. M2936) were seeded with 12,000 HeLa cells per well (ATCC Catalog No. CCL-2) in standard culture media containing DMEM (Thermo Fisher Catalog No. 11-965-092) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Catalog No. A4766) v/v, 1× GlutaMAX™ (Thermo Fisher Catalog No. 35050061) and 1× Penicillin-Streptomycin (Thermo Fisher Catalog No. 15140122). Following plating, cells were allowed to grow for 24 hours in an incubator at 37° C. with 5% CO₂. 25 ng of each gRNA fragment and 50 ng of the CRISPR-off plasmid (SEQ ID NO: 658) were resuspended in DPBS buffer (Thermo Fisher Catalog No. 14190144) to a concentration of 7.5 ng/μL. Additionally, 10 ng of EFla:PuromycinResistance plasmid was also added to the transfection mix to achieve a total payload of 85 ng of DNA. Transfection mixtures were created by adding the resuspended DNA components to Mirus® TransIT®-LT1 transfection reagent (Mirus Catalog No. MIR2300) following the manufacturer's instructions. 10 μL of each of the transfection mixtures was added in duplicate across a total of six screening plates. The positive controls used were CRISPRi (dCas9-KRAB) with two gRNAs targeting sites proximal to the TSS. The two CRISPRi positive control gRNAs used were gRNA004 and gRNA005 as annotated in the table of gRNA sequences. These control conditions are referred to as “CRISPRi-1” and “CRISPRi-2” respectively in the primary screen data tables. The negative controls were CRISPR-off without gRNA, CRISPR-off with a non-PCSK9 locus (CD151)-targeting gRNA, and empty vector (pUC19; NEB Catalog No. N3041S).

24 hours following transfection, a puromycin resistance selection was performed. The cell media was aspirated completely and all wells were washed 3× with DPBS buffer (Thermo Fisher Catalog No. 14190144) and 200 μL of 1 μg/μL puromycin was added to all screening plate wells.

48 hours following transfection, the cells were passaged. The cell media was completely aspirated and all wells were washed 3× with DPBS buffer (Thermo Fisher Catalog No. 14190144). Cells were enzymatically lifted by adding 25 μL of Trypsin-EDTA (0.25%) (Thermo Fisher Catalog No. 25200056) for five minutes in a 37° C. incubator. Trypsinized cells were resuspended 1:8 in fresh standard culture media and re-plated at a ratio of 1:4 72 hours after the media was changed.

In order to measure the level of secreted PCSK9 protein, media was harvested 24 hours after the media change and the cell plates were assayed for relative cell counts using the Promega Cell Titer Glo™ protocol (Catalog No. G7570) according to manufacturer's recommendations. PCSK9 protein levels were assessed using the LEGEND MAX™ Human PCSK9 ELISA Kit from BioLegend (Catalog No. 443107). Harvested media was plated and all subsequent steps were performed exactly according to manufacturer's recommendations. Final plate reads at 450 nm were performed on the Perkin Elmer® VICTOR® Nivo™ F instrument. GraphPad Prism software was used to fit a function to the standard curves and interpolate unknowns. PCSK9 ELISA results were normalized by Cell Titer Glo® assay (Promega Catalog No. G7571) results in order to correct for any well-to-well cell number variability.

Over 200 gRNAs were tested, of which 40 were identified as being top sequences (FIG. 2; top sequences designated as darker circles). The sequences and efficacies of the tested gRNAs are shown in Table 7 (SEQ: SEQ ID NO). The relative PCSK9 secretion (“% Control PCSK9”) represents the averaged PCSK9 protein levels of the treated samples expressed as a percent of the average across all non-targeting gRNA (CD151) negative control conditions. Robust silencing of PCSK9 (30-40% of negative control levels) was observed in cells treated with a number of gRNA candidate treatments. The top 40 gRNAs with the best PCSK9 protein knockdown were selected to be ordered as sgRNAs for further follow-up studies.

TABLE 7 Targeting Sequences of Tested gRNAs GRCh38 gRNA No. bp sequence % or to start Control Condition SEQ Targeting Sequence TSS coordinate PCSK9 CD151 N/A N/A N/A N/A 100 gRNA no gRNA N/A N/A N/A N/A 72.51 pUC N/A N/A N/A N/A 119.55 CRISPRi-1 N/A N/A N/A N/A 86.89 CRISPRi-2 N/A N/A N/A N/A 64.09 gRNA001 1262 GGUGCUAGCCUUGCGUUCCG 431 55039960 43.85 gRNA002 1263 CUGGCCGAAGCACCCGAGCA 462 55039991 74.87 gRNA003 1264 UGCGGAAACCUUCUAGGGUG 0 55039529 27.33 gRNA004 1265 GCGGAAACCUUCUAGGGUGU −1 55039528 34.93 gRNA005 1266 UCAAGCACCCACACCCUAGA −11 55039518 41.44 gRNA006 1267 GGGUGUGGGUGCUUGACGCC −15 55039514 42.41 gRNA007 1268 GGUGUGGGUGCUUGACGCCU −16 55039513 31.11 gRNA008 1269 GUGUGGGUGCUUGACGCCUG −17 55039512 36.13 gRNA009 1270 ACUGCCUGGCUCACUCCUCC 81 55039610 21.34 gRNA010 1271 UCACGCCACCAGAGCCCCAU −48 55039481 52.20 gRNA011 1272 AUCGUCCGAUGGGGCUCUGG −56 55039473 31.00 gRNA012 1273 AGGAUCGUCCGAUGGGGCUC −59 55039470 38.05 gRNA013 1274 UCAGAUAGGAUCGUCCGAUG −65 55039464 41.70 gRNA014 1275 GCGGCUCCCAGCUCCCAGCC 140 55039669 85.56 gRNA015 1276 CGGAAUCCUGGCUGGGAGCU 149 55039678 43.48 gRNA016 1277 GCGGAAUCCUGGCUGGGAGC 150 55039679 75.20 gRNA017 1278 GGGCGCGCGGAAUCCUGGCU 156 55039685 53.76 gRNA018 1279 GGGGCGCGCGGAAUCCUGGC 157 55039686 56.46 gRNA019 1280 UGAGGUCUUUGCAAACAAAG −919 55038610 76.59 gRNA020 1281 CCAGCACCUAGAUUCAGAGC −875 55038654 60.05 gRNA021 1282 CCUGCUCUGAAUCUAGGUGC −872 55038657 56.48 gRNA022 1283 UCGAUACUGGGAAGAAACAA −798 55038731 67.82 gRNA023 1284 CUGGAAGGGCUGUCGAUACU −786 55038743 59.17 gRNA024 1285 UCUGGAAGGGCUGUCGAUAC −785 55038744 63.62 gRNA025 1286 GAGGCUUGCUCUUUCUGGAA −772 55038757 58.91 gRNA026 1287 UGAGGCUUGCUCUUUCUGGA −771 55038758 74.33 gRNA027 1288 GACAUGAGGCUUGCUCUUUC −767 55038762 87.93 gRNA028 1289 UGUACAUGUGGCAUGACAUG −753 55038776 68.47 gRNA029 1290 CAUGCCACAUGUACAAUCUG −748 55038781 88.26 gRNA030 1291 CACAUGUACAAUCUGAGGCC −743 55038786 79.41 gRNA031 1292 CUGGCCUCAGAUUGUACAUG −741 55038788 106.36 gRNA032 1293 AAAAGGGGAAAGAGAGCUCC −722 55038807 91.18 gRNA033 1294 CCAGGCAGGAGGAUGAAAAG −707 55038822 89.34 gRNA034 1295 ACCAGGCAGGAGGAUGAAAA −706 55038823 79.11 gRNA035 1296 UACCAGGCAGGAGGAUGAAA −705 55038824 82.01 gRNA036 1297 CCUCCUGCCUGGUACACAAU −699 55038830 69.28 gRNA037 1298 CCUAUUGUGUACCAGGCAGG −696 55038833 82.57 gRNA038 1299 ACACCUAUUGUGUACCAGGC −693 55038836 58.95 gRNA039 1300 GGUACACAAUAGGUGUUUAC −689 55038840 81.55 gRNA040 1301 GUAAACACCUAUUGUGUACC −689 55038840 89.49 gRNA041 1302 UCCAGUUGAUUUCUUGAACA −659 55038870 69.65 gRNA042 1303 ACCAUGUUCAAGAAAUCAAC −655 55038874 73.51 gRNA043 1304 UCUUGAACAUGGUGUGUAAA −648 55038881 86.96 gRNA044 1305 UCUUUGCAAAUUGAAUCUUC −623 55038906 80.70 gRNA045 1306 AGUUUGCAAAGACGUCAUAU −559 55038970 85.13 gRNA046 1307 GAUUUAUACUACAAAGAUUC −531 55038998 74.88 gRNA047 1308 AGUUGGUAAGGUCAGUGUGC −454 55039075 66.05 gRNA048 1309 GUUGGUAAGGUCAGUGUGCA −453 55039076 60.64 gRNA049 1310 GCAGGGUGCAUAAAGGGCAG −436 55039093 60.72 gRNA050 1311 GGUGCAUAAAGGGCAGAGGC −432 55039097 92.12 gRNA051 1312 GCAUAAAGGGCAGAGGCCGG −429 55039100 53.71 gRNA052 1313 UUUAGAAGGCUGCCAGGUUA −392 55039137 56.64 gRNA053 1314 GCCCACCGAAUUCUUUCCAC −366 55039163 62.84 gRNA054 1315 AAAGAAUUCGGUGGGCAGCG −362 55039167 78.78 gRNA055 1316 CUUCUGAAUCAAUCCUACUG −333 55039196 52.04 gRNA056 1317 CUGGUCAGCAGGAGACAAGG −328 55039200 90.01 gRNA057 1318 GAUUGAUUCAGAAGUCUCAC −327 55039202 64.68 gRNA058 1319 CAGAAGUCUCACUGGUCAGC −319 55039210 101.68 gRNA059 1320 UCACUGGUCAGCAGGAGACA −311 55039218 75.12 gRNA060 1321 GCAGGAGACAAGGUGGACCC −301 55039228 43.48 gRNA061 1322 GGACCCAGGAAACACUGAAA −287 55039242 50.45 gRNA062 1323 CCCAGGAAACACUGAAAAGG −284 55039245 104.49 gRNA063 1324 CCAGGAAACACUGAAAAGGU −283 55039246 74.99 gRNA064 1325 AAACACUGAAAAGGUGGGCC −278 55039251 78.05 gRNA065 1326 UGGAGUCUGGCAUCCCACGC −248 55039281 86.50 gRNA066 1327 GGAGUCUGGCAUCCCACGCA −247 55039282 64.96 gRNA067 1328 CGGGAGAGGAGGAGCCCCUA −217 55039312 51.16 gRNA068 1329 AGGAGGAGCCCCUAGGGCGC −211 55039318 49.85 gRNA069 1330 AAGGCAGGCCGGCGCCCUAG −200 55039329 57.25 gRNA070 1331 GAAGGCAGGCCGGCGCCCUA −199 55039330 71.39 gRNA071 1332 GGAAGGCAGGCCGGCGCCCU −198 55039331 55.58 gRNA072 1333 AACUGGGCUGGAAGGCAGGC −189 55039340 81.36 gRNA073 1334 GCCUGCCUUCCAGCCCAGUU −189 55039340 68.77 gRNA074 1335 UCCUAACUGGGCUGGAAGGC −185 55039344 67.81 gRNA075 1336 CUUCCAGCCCAGUUAGGAUU −183 55039346 58.27 gRNA076 1337 UUCCAGCCCAGUUAGGAUUU −182 55039347 53.06 gRNA077 1338 CAAAUCCUAACUGGGCUGGA −181 55039348 31.55 gRNA078 1339 CUCCCAAAUCCUAACUGGGC −177 55039352 52.30 gRNA079 1340 AAAACUCCCAAAUCCUAACU −173 55039356 52.38 gRNA080 1341 AAAAACUCCCAAAUCCUAAC −172 55039357 67.62 gRNA081 1342 AGCGUCAGAUUACGCGCAGA −145 55039384 58.52 gRNA082 1343 CAGCGUCAGAUUACGCGCAG −144 55039385 76.23 gRNA083 1344 GCGCGUAAUCUGACGCUGUU −142 55039387 73.94 gRNA084 1345 CGCGUAAUCUGACGCUGUUU −141 55039388 71.95 gRNA085 1346 GCGUAAUCUGACGCUGUUUG −140 55039389 77.63 gRNA086 1347 UAAUCUGACGCUGUUUGGGG −137 55039392 70.70 gRNA087 1348 AAUCUGACGCUGUUUGGGGA −136 55039393 48.40 gRNA088 1349 GACGCUGUUUGGGGAGGGCG −131 55039398 64.83 gRNA089 1350 CGAAACCUGAUCCUCCAGUC −107 55039422 56.51 gRNA090 1351 GAAACCUGAUCCUCCAGUCC −106 55039423 45.68 gRNA091 1352 AAACCUGAUCCUCCAGUCCG −105 55039424 50.44 gRNA092 1353 CGGACUGGAGGAUCAGGUUU −105 55039424 30.88 gRNA093 1354 AACCUGAUCCUCCAGUCCGG −104 55039425 29.56 gRNA094 1355 AACCCCCGGACUGGAGGAUC −99 55039430 33.66 gRNA095 1356 UAACGGAACCCCCGGACUGG −93 55039436 35.57 gRNA096 1357 CAUUAACGGAACCCCCGGAC −90 55039439 30.87 gRNA097 1358 UUAAACAUUAACGGAACCCC −85 55039444 58.22 gRNA098 1359 CCGUUAAUGUUUAAUCAGAU −79 55039450 58.99 gRNA099 1360 CCUAUCUGAUUAAACAUUAA −76 55039453 45.83 gRNA100 1361 AAUCAGAUAGGAUCGUCCGA −67 55039462 47.05 gRNA101 1362 AUCAGAUAGGAUCGUCCGAU −66 55039463 57.76 gRNA102 1363 UGGCGUGAUCUGCGCGCCCC −36 55039493 41.66 gRNA103 1364 GUCGCUGCGGAAACCUUCUA 5 55039534 52.14 gRNA104 1365 CGUCGCUGCGGAAACCUUCU 6 55039535 54.97 gRNA105 1366 AGGUUUCCGCAGCGACGUCG 9 55039538 61.69 gRNA106 1367 CAGCGACGUCGAGGCGCUCA 18 55039547 54.52 gRNA107 1368 UGAGCGCCUCGACGUCGCUG 18 55039547 35.11 gRNA108 1369 GUCGAGGCGCUCAUGGUUGC 25 55039554 25.10 gRNA109 1370 GAGGCGCUCAUGGUUGCAGG 28 55039557 43.19 gRNA110 1371 AGGCGCUCAUGGUUGCAGGC 29 55039558 29.00 gRNA111 1372 AGUUCAGGGUCUGAGCCUGG 43 55039571 39.04 gRNA112 1373 CGGGCGCCGCCGUUCAGUUC 48 55039577 45.13 gRNA113 1374 GGGCGCCGCCGUUCAGUUCA 49 55039578 32.30 gRNA114 1375 UCAGACCCUGAACUGAACGG 57 55039586 55.95 gRNA115 1376 GGCUCAGACCCUGAACUGAA 60 55039589 48.59 gRNA116 1377 UUCAGUUCAGGGUCUGAGCC 60 55039589 57.38 gRNA117 1378 UGAGCCUGGAGGAGUGAGCC 74 55039603 45.58 gRNA118 1379 AGUGAGCCAGGCAGUGAGAC 86 55039615 46.98 gRNA119 1380 GCCAGGCAGUGAGACUGGCU 91 55039620 62.91 gRNA120 1381 CCAGGCAGUGAGACUGGCUC 92 55039621 54.17 gRNA121 1382 CCCGAGCCAGUCUCACUGCC 95 55039624 48.29 gRNA122 1383 GGCAGUGAGACUGGCUCGGG 95 55039624 34.80 gRNA123 1384 GCAGUGAGACUGGCUCGGGC 96 55039625 53.96 gRNA124 1385 UGAGACUGGCUCGGGCGGGC 100 55039629 59.99 gRNA125 1386 GAGACUGGCUCGGGCGGGCC 101 55039630 34.59 gRNA126 1387 GGGACGCGUCGUUGCAGCAG 121 55039650 38.46 gRNA127 1388 GCUGCUGCAACGACGCGUCC 122 55039651 44.41 gRNA128 1389 UGAAGGGGCGCGCGGAAUCC 161 55039690 28.00 gRNA129 1390 AGGGCGCGUGAAGGGGCGCG 169 55039698 64.66 gRNA130 1391 CAGGAGCAGGGCGCGUGAAG 176 55039705 52.46 gRNA131 1392 UCAGGAGCAGGGCGCGUGAA 177 55039706 61.68 gRNA132 1393 UUCAGGAGCAGGGCGCGUGA 178 55039707 48.06 gRNA133 1394 GGAGCUGAAGUUCAGGAGCA 188 55039717 62.68 gRNA134 1395 AGGAGCUGAAGUUCAGGAGC 189 55039718 66.77 gRNA135 1396 CUGUGCAGGAGCUGAAGUUC 195 55039724 47.35 gRNA136 1397 GCACAGUCCUCCCCACCGCA 209 55039738 40.95 gRNA137 1398 UGCGGUGGGGAGGACUGUGC 209 55039738 51.32 gRNA138 1399 CCUCCCCACCGCAAGGCUCA 216 55039745 76.44 gRNA139 1400 CCUUGAGCCUUGCGGUGGGG 219 55039748 71.95 gRNA140 1401 GCGCCUUGAGCCUUGCGGUG 222 55039751 63.12 gRNA141 1402 GGCGCCUUGAGCCUUGCGGU 223 55039752 64.08 gRNA142 1403 CGGCGCCUUGAGCCUUGCGG 224 55039753 68.16 gRNA143 1404 CGCAAGGCUCAAGGCGCCGC 225 55039754 58.50 gRNA144 1405 GGCUCAAGGCGCCGCCGGCG 230 55039759 41.31 gRNA145 1406 AGGCCGUGCGCGGUCCACGC 247 55039776 46.31 gRNA146 1407 GUGGACCGCGCACGGCCUCU 249 55039778 52.25 gRNA147 1408 GGAGACCUAGAGGCCGUGCG 257 55039786 62.38 gRNA148 1409 CAGGACAGCAACCUCUCCCC 281 55039810 59.44 gRNA149 1410 UGGGCACCGUCAGCUCCAGG 310 55039839 54.56 gRNA150 1411 CCGUCAGCUCCAGGCGGUCC 316 55039845 47.26 gRNA151 1412 CCAGGACCGCCUGGAGCUGA 319 55039848 71.17 gRNA152 1413 UCAGCUCCAGGCGGUCCUGG 319 55039848 46.68 gRNA153 1414 CAGCGGCCACCAGGACCGCC 328 55039857 67.26 gRNA154 1415 CUGCUGCUCCUGGGUCCCGC 366 55039895 54.47 gRNA155 1416 CACGGGCGCCCGCGGGACCC 377 55039906 48.18 gRNA156 1417 UCCCGCGGGCGCCCGUGCGC 380 55039909 53.18 gRNA157 1418 CGCGGGCGCCCGUGCGCAGG 383 55039912 61.58 gRNA158 1419 UCCUGCGCACGGGCGCCCGC 384 55039913 67.30 gRNA159 1420 CUCCUGCGCACGGGCGCCCG 385 55039914 48.64 gRNA160 1421 CGCCCGUGCGCAGGAGGACG 389 55039918 84.11 gRNA161 1422 CGUGCGCAGGAGGACGAGGA 393 55039922 86.55 gRNA162 1423 GUCCUCGUCCUCCUGCGCAC 394 55039923 54.45 gRNA163 1424 CGUCCUCGUCCUCCUGCGCA 395 55039924 80.30 gRNA164 1425 GGACGAGGACGGCGACUACG 404 55039933 69.15 gRNA165 1426 GGACGGCGACUACGAGGAGC 410 55039939 62.54 gRNA166 1427 GCUAGCCUUGCGUUCCGAGG 434 55039963 60.30 gRNA167 1428 GCCUUGCGUUCCGAGGAGGA 438 55039967 57.71 gRNA168 1429 GCCGUCCUCCUCGGAACGCA 442 55039971 62.04 gRNA169 1430 GCGUUCCGAGGAGGACGGCC 443 55039972 79.68 gRNA170 1431 UUCGGCCAGGCCGUCCUCCU 451 55039980 40.06 gRNA171 1432 CGUGCUCGGGUGCUUCGGCC 464 55039993 59.35 gRNA172 1433 GUGGCUGUGGUUCCGUGCUC 477 55040006 45.91 gRNA173 1434 GGUGGCUGUGGUUCCGUGCU 478 55040007 70.81 gRNA174 1435 GCAGCGGUGGAAGGUGGCUG 490 55040019 72.70 gRNA175 1436 CACCUUCCACCGCUGCGCCA 494 55040023 86.51 gRNA176 1437 CUUGGCGCAGCGGUGGAAGG 496 55040025 76.28 gRNA177 1438 CACCUUGGCGCAGCGGUGGA 499 55040028 66.94 gRNA178 1439 UCCACCGCUGCGCCAAGGUG 499 55040028 48.71 gRNA179 1440 CCACCGCUGCGCCAAGGUGC 500 55040029 60.24 gRNA180 1441 CCCGCACCUUGGCGCAGCGG 503 55040032 44.64 gRNA181 1442 GGGCGAACCCGCAGCCGGGA 541 55040070 46.89 gRNA182 1443 UCCCGGCUGCGGGUUCGCCC 541 55040070 57.20 gRNA183 1444 CACCGCACCGUCCCGGCUGC 551 55040080 76.92 gRNA184 1445 GCACCGCACCGUCCCGGCUG 552 55040081 85.00 gRNA185 1446 GAAACAGCACCGCACCGUCC 558 55040087 75.31 gRNA186 1447 GGUGCGGUGCUGUUUCCUCU 562 55040091 51.49 gRNA187 1448 GUGCGGUGCUGUUUCCUCUC 563 55040092 57.43 gRNA188 1449 GGGGGAAACUGAGGCCCGAG 577 55040105 55.16 gRNA189 1450 AGUUUCCCCCCAUGUAAGAG 590 55040119 45.91 gRNA190 1451 CCCCCAUGUAAGAGAGGAAG 596 55040125 63.89 gRNA191 1452 CACUUCCUCUCUUACAUGGG 598 55040127 63.57 gRNA192 1453 CCACUUCCUCUCUUACAUGG 599 55040128 66.47 gRNA193 1454 UAAGAGAGGAAGUGGAGUGC 604 55040133 63.94 gRNA194 1455 GUCGCCGAGGGCUCUUCGCU 626 55040155 55.19 gRNA195 1456 CGUGCCAAGCGAAGAGCCCU 633 55040162 66.66 gRNA196 1457 CUCUUCGCUUGGCACGAUCU 637 55040166 57.10 gRNA197 1458 UCUUCGCUUGGCACGAUCUU 638 55040167 44.24 gRNA198 1459 CUUCGCUUGGCACGAUCUUG 639 55040168 50.40 gRNA199 1460 GGCACGAUCUUGGGGACUGC 647 55040176 73.29 gRNA200 1461 GAUCUUGGGGACUGCAGGCA 652 55040181 56.39 gRNA201 1462 CUUGGGGACUGCAGGCAAGG 655 55040184 66.89 gRNA202 1463 GGACUGCAGGCAAGGCGGCG 660 55040189 61.44 gRNA203 1464 GACUGCAGGCAAGGCGGCGG 661 55040190 49.55 gRNA204 1465 UGCAGGCAAGGCGGCGGGGG 664 55040193 41.85 gRNA205 1466 GGCAAGGCGGCGGGGGAGGA 668 55040197 95.50 gRNA206 1467 GCAAGGCGGCGGGGGAGGAC 669 55040198 120.71 gRNA207 1468 GUGGGGAGCACGGUGGAGAG 694 55040223 73.17 gRNA208 1469 UGGGGAGCACGGUGGAGAGC 695 55040224 61.04 gRNA209 1470 GGGGAGCACGGUGGAGAGCG 696 55040225 79.49 gRNA210 1471 AGCACGGUGGAGAGCGGGGA 700 55040229 42.81 gRNA211 1472 CGGUGGAGAGCGGGGACGGC 704 55040233 79.46 gRNA212 1473 CGUGCGGCUGCGCUAUUCAG 748 55040277 39.48 gRNA213 1474 GUGCGGCUGCGCUAUUCAGU 749 55040278 56.25 gRNA214 1475 GGCUGCGCUAUUCAGUGGGA 753 55040282 82.51 gRNA215 1476 CUAUUCAGUGGGAAGGUUCG 760 55040289 77.53 gRNA216 1477 UAUUCAGUGGGAAGGUUCGC 761 55040290 71.30 gRNA217 1478 AUUCAGUGGGAAGGUUCGCG 762 55040291 68.82 gRNA218 1479 AGUGGGAAGGUUCGCGGGGU 766 55040295 73.55 gRNA219 1480 GUGGGAAGGUUCGCGGGGUU 767 55040296 66.13 gRNA220 1481 AGGGCGAGCAGAGCACUGCC 808 55040337 68.85 gRNA221 1482 UUUCUGCCUCGCCGCGGCAC 853 55040382 65.59 gRNA222 1483 CUGCCUCGCCGCGGCACAGG 856 55040385 76.58 gRNA223 1484 UGCCUCGCCGCGGCACAGGU 857 55040386 75.75 gRNA224 1485 CACCCACCUGUGCCGCGGCG 862 55040391 81.79 gRNA225 1486 UCCUUCACCCACCUGUGCCG 867 55040396 70.86 gRNA226 1487 UGGGUGAAGGAGUGAAUGCC 876 55040405 57.69

Best-performing gRNAs were found to closely align to the PCSK9 gene transcription start site (FIG. 3).

Following this primary screen, a secondary screen was performed with the top 40 gRNAs in RNA form. The top 40 guides were chemically synthesized and co-transfected with in vitro transcribed mRNA encoding the CRISPR-off, CRISPRi or WT Cas9 constructs. Secreted PCSK9 levels were measured 7 and 28 days after transfection.

To generate in vitro transcribed CRISPR-off, CRISPRi and WT Cas9 effector mRNA, plasmid constructs encoding these proteins were linearized using MfeI restriction enzyme from NEB® (Catalog No. R3589S). 1 g of linearized template was used to set up in vitro transcription reactions using T7 mScript™ Standard mRNA Production System from CellScript (Catalog No. C-MSC100625) according to manufacturer's instructions. The resulting RNA had a Cap 1 structure on the 5′ end and was 3′ polyadenlylated. The transcribed RNA was purified using the RNeasy® Mini Kit from Qiagen (Catalog No. 74104).

End-modified sgRNAs purified using standard desalting were obtained from Integrated DNA Technologies. The three nucleotides at the 5′ end and the three nucleotides at the 3′ end of each guide were 2′-O-methyl modified. The three internucleoside linkages at the 3′ end and the three internucleoside linkages at the 5′ end were phosphorothioate internucleoside linkages (Table 8; SEQ: SEQ ID NO). In the Table, mX (i.e., mA, mC, mG, or mU) represents a 2′-O-methyl modified ribonucleoside, rX (i.e., rA, rC, rG, or rU) indicates a natural ribonucleoside, and * indicates a phosphorothioate linkage. All internucleoside linkages that are not phosphorothioate linkages are phosphate linkages.

TABLE 8 Targeting Sequences of Top 40 gRNAs from Secondary HeLa Cell Screen gRNA Targeting Modified Full Modified RNA No. SEQ Sequence gRNA No. Targeting Sequence gRNA 1270 ACUGCCUGGCU modRNA009 mA*mC*mU*rGrCrCrUrGrGrCrUrCr 009 CACUCCUCC ArCrUrCrCrUrCrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1369 GUCGAGGCGCU modRNA108 mG*mU*mC*rGrArGrGrCrGrCrUrCr 108 CAUGGUUGC ArUrGrGrUrUrGrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1264 UGCGGAAACCU modRNA003 mU*mG*mC*rGrGrArArArCrCrUrUr 003 UCUAGGGUG CrUrArGrGrGrUrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1389 UGAAGGGGCGC modRNA128 mU*mG*mA*rArGrGrGrGrCrGrCrGr 128 GCGGAAUCC CrGrGrArArUrCrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1371 AGGCGCUCAUG modRNA110 mA*mG*mG*rCrGrCrUrCrArUrGrGr 110 GUUGCAGGC UrUrGrCrArGrGrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1354 AACCUGAUCCU modRNA093 mA*mA*mC*rCrUrGrArUrCrCrUrCr 093 CCAGUCCGG CrArGrUrCrCrGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1357 CAUUAACGGAA modRNA096 mC*mA*mU*rUrArArCrGrGrArArCr 096 CCCCCGGAC CrCrCrCrGrGrArCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1353 CGGACUGGAGG modRNA092 mC*mG*mG*rArCrUrGrGrArGrGrAr 092 AUCAGGUUU UrCrArGrGrUrUrUrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1272 AUCGUCCGAUG modRNA011 mA*mU*mC*rGrUrCrCrGrArUrGrGr 011 GGGCUCUGG GrGrCrUrCrUrGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1268 GGUGUGGGUGC modRNA007 mG*mG*mU*rGrUrGrGrGrUrGrCrUr 007 UUGACGCCU UrGrArCrGrCrCrUrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1338 CAAAUCCUAAC modRNA077 mC*mA*mA*rArUrCrCrUrArArCrUr 077 UGGGCUGGA GrGrGrCrUrGrGrArGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1374 GGGCGCCGCCG modRNA113 mG*mG*mG*rCrGrCrCrGrCrCrGrUr 113 UUCAGUUCA UrCrArGrUrUrCrArGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1355 AACCCCCGGAC modRNA094 mA*mA*mC*rCrCrCrCrGrGrArCrUr 094 UGGAGGAUC GrGrArGrGrArUrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1386 GAGACUGGCUC modRNA125 mG*mA*mG*rArCrUrGrGrCrUrCrGr 125 GGGCGGGCC GrGrCrGrGrGrCrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1383 GGCAGUGAGAC modRNA122 mG*mG*mC*rArGrUrGrArGrArCrUr 122 UGGCUCGGG GrGrCrUrCrGrGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1265 GCGGAAACCUU modRNA004 mG*mC*mG*rGrArArArCrCrUrUrCr 004 CUAGGGUGU UrArGrGrGrUrGrUrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1368 UGAGCGCCUCG modRNA107 mU*mG*mA*rGrCrGrCrCrUrCrGrAr 107 ACGUCGCUG CrGrUrCrGrCrUrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1356 UAACGGAACCC modRNA095 mU*mA*mA*rCrGrGrArArCrCrCrCr 095 CCGGACUGG CrGrGrArCrUrGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1269 GUGUGGGUGCU modRNA008 mG*mU*mG*rUrGrGrGrUrGrCrUrUr 008 UGACGCCUG GrArCrGrCrCrUrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1273 AGGAUCGUCCG modRNA012 mA*mG*mG*rArUrCrGrUrCrCrGrAr 012 AUGGGGCUC UrGrGrGrGrCrUrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1387 GGGACGCGUCG modRNA126 mG*mG*mG*rArCrGrCrGrUrCrGrUr 126 UUGCAGCAG UrGrCrArGrCrArGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1372 AGUUCAGGGUC modRNA111 mA*mG*mU*rUrCrArGrGrGrUrCrUr 111 UGAGCCUGG GrArGrCrCrUrGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1473 CGUGCGGCUGC modRNA212 mC*mG*mU*rGrCrGrGrCrUrGrCrGr 212 GCUAUUCAG CrUrArUrUrCrArGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1431 UUCGGCCAGGC modRNA170 mU*mU*mC*rGrGrCrCrArGrGrCrCr 170 CGUCCUCCU GrUrCrCrUrCrCrUrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1397 GCACAGUCCUC modRNA136 mG*mC*mA*rCrArGrUrCrCrUrCrCr 136 CCCACCGCA CrCrArCrCrGrCrArGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1405 GGCUCAAGGCG modRNA144 mG*mG*mC*rUrCrArArGrGrCrGrCr 144 CCGCCGGCG CrGrCrCrGrGrCrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1266 UCAAGCACCCA modRNA005 mU*mC*mA*rArGrCrArCrCrCrArCr 005 CACCCUAGA ArCrCrCrUrArGrArGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1363 UGGCGUGAUCU modRNA102 mU*mG*mG*rCrGrUrGrArUrCrUrGr 102 GCGCGCCCC CrGrCrGrCrCrCrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1274 UCAGAUAGGAU modRNA013 mU*mC*mA*rGrArUrArGrGrArUrCr 013 CGUCCGAUG GrUrCrCrGrArUrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1465 UGCAGGCAAGG modRNA204 mU*mG*mC*rArGrGrCrArArGrGrCr 204 CGGCGGGGG GrGrCrGrGrGrGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1267 GGGUGUGGGUG modRNA006 mG*mG*mG*rUrGrUrGrGrGrUrGrCr 006 CUUGACGCC UrUrGrArCrGrCrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1471 AGCACGGUGGA modRNA210 mA*mG*mC*rArCrGrGrUrGrGrArGr 210 GAGCGGGGA ArGrCrGrGrGrGrArGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1370 GAGGCGCUCAU modRNA109 mG*mA*mG*rGrCrGrCrUrCrArUrGr 109 GGUUGCAGG GrUrUrGrCrArGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1321 GCAGGAGACAA modRNA060 mG*mC*mA*rGrGrArGrArCrArArGr 060 GGUGGACCC GrUrGrGrArCrCrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1276 CGGAAUCCUGG modRNA015 mC*mG*mG*rArArUrCrCrUrGrGrCr 015 CUGGGAGCU UrGrGrGrArGrCrUrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1262 GGUGCUAGCCU modRNA001 mG*mG*mU*rGrCrUrArGrCrCrUrUr 001 UGCGUUCCG GrCrGrUrUrCrCrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1458 UCUUCGCUUGG modRNA197 mU*mC*mU*rUrCrGrCrUrUrGrGrCr 197 CACGAUCUU ArCrGrArUrCrUrUrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1388 GCUGCUGCAAC modRNA127 mG*mC*mU*rGrCrUrGrCrArArCrGr 127 GACGCGUCC ArCrGrCrGrUrCrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1441 CCCGCACCUUG modRNA180 mC*mC*mC*rGrCrArCrCrUrUrGrGr 180 GCGCAGCGG CrGrCrArGrCrGrGrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU gRNA 1373 CGGGCGCCGCC modRNA112 mC*mG*mG*rGrCrGrCrCrGrCrCrGr 112 GUUCAGUUC UrUrCrArGrUrUrCrGrUrUrUrArArGr ArGrCrUrArArGrCrUrGrGrArArArCr ArGrCrArUrArGrCrArArGrUrUrUrAr ArArUrArArGrGrCrUrArGrUrCrCrGr UrUrArUrCrArArCrUrUrGrArArArAr ArGrUrGrGrCrArCrCrGrArGrUrCrGr GrUrGrCrUrUrU*mU*mU*mU

HeLa cells were reverse transfected with 25 ng effector and 12.5 ng sgRNA in a 96 well plate format using TransIT®-X2 transfection reagent from Minis (Catalog No. MIR6003). Conditioned media was harvested every week for up to four weeks and used to measure secreted PCSK9 levels using LEGEND MAX™ Human PCSK9 ELISA Kit from BioLegend (Catalog No. 443107). ELISA data were normalized for cell numbers using the CellTiter-Glo® kit from Promega (Catalog No. G7571). While PCSK9 silencing was transient with CRISPRi (dCas9-KRAB), and returned to baseline by day 28, several sgRNAs co-transfected with CRISPR-off (DNMT3A-3L-dCas9-KRAB) construct showed robust and durable silencing (FIG. 4A). 16 out of the 40 guides tested with CRISPR-off showed silencing efficiency greater than WT Cas9 (Table 9).

TABLE 9 Relative PCSK9 Expression in HeLa Cells Treated with Modified gRNAs and CRISPR-off D 28, % of control Condition (Average) Negative Controls Transfection reagent only 100 CRISPRi IVT + modRNA009 108.1 CRISPRi IVT + modRNA004 88.1 Positive Control WT Cas9 IVT + modRNA180 10.5 Experimental Conditions (CRISPR-off and indicated modified gRNA) modRNA009 5.7 modRNA108 12.9 modRNA003 7.5 modRNA128 16.2 modRNA110 5.7 modRNA093 11.7 modRNA096 20.7 modRNA092 12.7 modRNA011 9.6 modRNA007 11.6 modRNA077 4.2 modRNA113 4.7 modRNA094 12.0 modRNA125 67.4 modRNA122 9.5 modRNA004 1.0 modRNA107 7.7 modRNA095 30.5 modRNA008 4.7 modRNA012 6.0 modRNA126 6.1 modRNA111 4.0 modRNA212 25.7 modRNA170 14.8 modRNA136 17.2 modRNA144 35.0 modRNA005 7.9 modRNA102 25.7 modRNA013 5.2 modRNA204 64.4 modRNA006 36.6 modRNA210 30.0 modRNA109 25.3 modRNA060 33.3 modRNA015 19.4 modRNA001 18.2 modRNA197 45.1 modRNA127 13.4 modRNA180 30.2 modRNA112 6.1

At the two-week time point, RNA was extracted using Quick-RNA 96 Kit from Zymo Research (Cat #R1053). qPCR was performed using qScript XLT One-Step RT-qPCR ToughMix from Quantabio (Cat #95134-500) and TaqMan assays (PCSK9: Hs00545399_ml, PPIA: Hs99999904_ml). PCSK9 levels were normalized with PPIA. Relative quantification was done using delta-delta Ct method.

Suppression of PCSK9 secretion was found to correlate with mRNA silencing at day 14 (FIG. 4B).

modRNA004 and modRNA111 were tested in HeLa cells for suppressing PCSK9 secretion over 60 days (FIG. 5). WT Cas9 was co-transfected with modRNA180 as a positive control. Cells were treated with 25 ng of the effector and 12.5 ng of the gRNA. modRNA004 and modRNA111 were shown to mediate durable silencing of PCSK9, comparable to what was achieved by WT Cas9 in HeLa cells via gene editing.

Furthermore, results suggest that epigenetic silencing is maintained in HeLa cells treated with simvastatin. Statin treatment is known to increase PCSK9 secretion via a transcriptional mechanism. In epigenetically silenced HeLa cells, statin treatment was shown to not increase PCSK9 secretion (FIG. 6).

Example 5: Guide RNA Assays in Huh7 Hepatoma Cell Line

The Huh7 hepatoma cell line is amenable to high-throughput screening and transfection. The top 13 guides from the HeLa screen that had either perfect homology or a single mismatch with the cynomolgus PCSK9 gene were tested in Huh7 hepatoma cell line. Epigenetic silencing with the CRISPR-off construct was shown to be stable over seven days (FIG. 7).

Example 6: Guide RNA Assays in Primary Human and Cynomolgus Hepatocytes

Primary human and cynomolgus HepatoPac® cultures from BioIVT are used to test the efficacy of the gRNAs in primary hepatocytes. HepatoPac® cultures are maintained according to manufacturer recommendations. Briefly, HepatoPac® maintenance media is thawed and made up within 30 minutes of cells arrival. Upon media change, cells are allowed to acclimate for two days in 37° C., 10% CO₂incubator. On the second day after receipt, the manufacturer's instructions are followed to formulate LNP's with CRISPR-off+sgRNA, GFP-mRNA and WT CRISPR Cas9 in various concentrations using the SPARK™ (Precision Nanosystems) and the hepato9 mRNA LNP formulation kit (CAT. Number NWS0016). Subsequently, the LNP's are characterized for encapsulation efficiency and total mRNA payload delivery via the Quant-it™ RiboGreen RNA assay kit. LNPs are then added to the media in specified quantities of total mRNA to achieve clinically relevant levels of silencing of PCSK9. Media is changed every other day for a duration of up to four weeks to assess durability and/or inheritability of silencing. PCSK9 silencing will be assessed every seven days by ELISA to measure secreted PCSK9 levels in the media. PCSK9 concentration will be controlled to total hepatocytes using a human albumin ELISA (Thermo Fisher®). Data is then presented as total PCSK9 secretion as percent of GFP-mRNA negative control. Specificity may be assessed by isolating the primary human and cynomolgus hepatocytes from the mouse fibroblast feeder layer using a magnetic bead based antibody approach (Miltenyi Biotec). Following separation of primary hepatocytes from the feeder layer, cells are processed for RNAseq evaluation and genome-wide bisulfite sequencing.

The top 13 gRNAs in RNA format are selected to be tested in primary human hepatocytes (PHH). The top gRNAs are selected based on (i) PCSK9 silencing efficiency and durability in HeLa cells (ii) whether they have a perfect alignment with the human PCSK9 gene and up to one mismatch with the non-human primate PCSK9 gene. Combinations of gRNAs are also tested to determine their efficacy and durability. The negative controls are CRISPR-off only, gRNA Fragment Only (modRNA003), and CRISPRi only. The positive control is CRISPRi co-transfected with modRNA004 (Table 10; SEQ: SEQ ID NO; NHP: non-human primate). All tested gRNAs are predicted to bind to both human and non-human primate PCSK9.

TABLE 10 gRNAs Being Screened in Primary Human Hepatocytes gRNA TSS Modified Match to No. SEQ gRNA Targeting Sequence distance RNA No. NHP gRNA009 1270 ACUGCCUGGCUCACUCCUCC 81 modRNA009 Exact gRNA003 1264 UGCGGAAACCUUCUAGGGUG 0 modRNA003 Exact gRNA093 1354 AACCUGAUCCUCCAGUCCGG −104 modRNA093 Exact gRNA011 1272 AUCGUCCGAUGGGGCUCUGG −56 modRNA011 Exact gRNA007 1268 GGUGUGGGUGCUUGACGCCU −16 modRNA007 Exact gRNA077 1338 CAAAUCCUAACUGGGCUGGA −181 modRNA077 1 mismatch gRNA113 1374 GGGCGCCGCCGUUCAGUUCA 49 modRNA113 1 mismatch gRNA004 1265 GCGGAAACCUUCUAGGGUGU −1 modRNA004 Exact gRNA008 1269 GUGUGGGUGCUUGACGCCUG −17 modRNA008 Exact gRNA012 1273 AGGAUCGUCCGAUGGGGCUC −59 modRNA012 Exact gRNA111 1372 AGUUCAGGGUCUGAGCCUGG 43 modRNA111 1 mismatch gRNA005 1266 UCAAGCACCCACACCCUAGA −11 modRNA005 Exact gRNA013 1274 UCAGAUAGGAUCGUCCGAUG −65 modRNA013 Exact

Robust PCSK9 silencing is observed. For some gRNAs, >70% reduction in secreted PCSK9 at day 7 is observed (depending on transfection efficiency).

Example 7: ZF Assays in HeLa Cells

A total of 209 zinc finger proteins (architecture as shown in SEQ ID NO: 659) were designed from the ZF library to 49 PCSK9 target sites (selected from GRCh38 chromosome 1 between 55038548 to 55040548). The target sites had no other exact matches in the human genome (GRCh38).

HeLa cells were transfected with ZF-off constructs in DNA format. Six 96-well plates (Sigma-Aldrich Catalog No. M2936) were seeded with 12,000 HeLa cells per well (ATCC Catalog No. CCL-2) in standard culture media containing DMEM (Thermo Fisher Catalog No. 11-965-092) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Catalog No. A4766) v/v, 1× GlutaMAX™ (Thermo Fisher Catalog No. 35050061) and 1× Penicillin-Streptomycin (Thermo Fisher Catalog No. 15140122). Following plating, cells were allowed to grow for 24 hours in a 37° C. incubator at 5% CO₂. 10 ng of the ZF-off plasmid was resuspended in DPBS buffer (Thermo Fisher Catalog No. 14190144) to a concentration of 7.5 ng/μL. In addition, 10 ng of EF1a:PuromycinResistance plasmid and 65 ng of empty vector (pUC19) were also added to the transfection mix to achieve a total payload of 85 ng of DNA. Transfection mixtures were created by adding resuspended DNA in serum-free OPTI-MEM media (Thermo Fisher® Catalog No. 31985062) and adding Mirus® TransIT®-LT1 transfection reagent (MIR2300) following the manufacturer's instructions. 10 μL of transfection mixtures were added in duplicate across a total of six screening plates. The positive control was CRISPR-off (SEQ ID NO: 658) with a high performing gRNA (gRNA009). Negative controls were ZF-off with a non-PCSK9 locus target (CLTA) and empty vector (pUC 19; NEB Catalog No. N3041 S).

The ZF screen yielded hits with activity comparable to CRISPR (FIG. 8). The candidates with high silencing efficiency are taken forward to follow-up experiments. FIG. 9 shows the ZF screening results by distance to TSS. In total, 209 ZFs were screened, with their PCSK9 knockdown activity relative to the negative control shown in Table 11 below.

TABLE 11 ZF-off Construct Activity ZF No. % Control ZF No. % Control ZF No. % Control ZF028 37.43 ZF071 66.51 ZF141 104.50 ZF001 35.90 ZF072 62.59 ZF142 71.70 ZF047 32.08 ZF073 69.50 ZF143 49.70 ZF048 15.19 ZF074 55.74 ZF144 44.01 ZF029 40.42 ZF075 51.58 ZF145 60.80 ZF030 7.74 ZF076 58.82 ZF146 42.85 ZF031 29.80 ZF077 49.52 ZF147 58.42 ZF032 40.65 ZF078 52.50 ZF148 70.50 ZF033 30.71 ZF079 66.30 ZF149 95.42 ZF034 31.95 ZF080 51.52 ZF150 74.00 ZF035 38.29 ZF081 140.85 ZF151 61.01 ZF002 33.02 ZF082 105.84 ZF152 52.34 ZF003 29.17 ZF083 72.30 ZF153 80.11 ZF004 35.39 ZF084 95.05 ZF154 79.46 ZF005 37.90 ZF085 44.18 ZF155 50.09 ZF006 37.47 ZF086 88.95 ZF156 46.99 ZF036 35.19 ZF087 44.87 ZF157 89.03 ZF037 28.76 ZF088 56.44 ZF158 99.89 ZF038 21.11 ZF089 116.41 ZF159 101.66 ZF039 9.35 ZF090 51.30 ZF160 119.21 ZF040 5.20 ZF091 57.46 ZF161 49.64 ZF041 30.50 ZF092 46.82 ZF162 68.98 ZF007 36.13 ZF093 44.03 ZF163 89.71 ZF008 34.72 ZF094 65.55 ZF164 103.15 ZF009 28.91 ZF095 56.92 ZF165 48.37 ZF010 33.58 ZF096 93.13 ZF166 81.13 ZF011 41.01 ZF097 57.08 ZF167 67.29 ZF012 19.39 ZF098 57.03 ZF168 50.88 ZF013 40.46 ZF099 59.36 ZF169 77.25 ZF014 33.06 ZF100 95.34 ZF170 108.08 ZF015 22.48 ZF101 57.13 ZF171 57.10 ZF016 34.09 ZF102 145.81 ZF172 53.73 ZF017 24.71 ZF103 63.08 ZF173 62.61 ZF018 24.84 ZF104 50.06 ZF174 71.84 ZF019 14.70 ZF105 72.80 ZF175 68.12 ZF020 33.15 ZF106 75.56 ZF176 51.52 ZF021 33.87 ZF107 56.29 ZF177 48.59 ZF022 29.02 ZF108 103.79 ZF178 71.10 ZF023 30.45 ZF109 60.41 ZF179 53.08 ZF024 12.55 ZF110 53.71 ZF180 54.56 ZF042 41.13 ZF111 69.30 ZF181 78.86 ZF025 39.37 ZF112 94.36 ZF182 70.20 ZF026 28.27 ZF113 57.16 ZF183 69.07 ZF043 40.92 ZF114 51.72 ZF184 49.73 ZF044 37.27 ZF115 78.55 ZF185 69.04 ZF045 39.72 ZF116 76.55 ZF186 57.09 ZF027 38.67 ZF117 66.49 ZF187 64.90 ZF046 36.12 ZF118 60.58 ZF188 59.91 ZF049 43.53 ZF119 78.70 ZF189 59.92 ZF050 44.02 ZF120 63.82 ZF190 79.85 ZF051 46.90 ZF121 117.94 ZF191 71.95 ZF052 45.89 ZF122 82.54 ZF192 78.47 ZF053 42.71 ZF123 84.94 ZF193 74.79 ZF054 44.31 ZF124 71.86 ZF194 67.87 ZF055 44.57 ZF125 76.73 ZF195 83.86 ZF056 44.82 ZF126 119.64 ZF196 67.71 ZF057 46.52 ZF127 144.15 ZF197 64.36 ZF058 46.61 ZF128 66.74 ZF198 62.06 ZF059 47.62 ZF129 86.03 ZF199 59.08 ZF060 44.75 ZF130 103.82 ZF200 53.09 ZF061 59.99 ZF131 116.46 ZF201 57.39 ZF062 48.69 ZF132 130.63 ZF202 63.53 ZF063 50.63 ZF133 100.63 ZF203 69.94 ZF064 54.60 ZF134 42.35 ZF204 67.61 ZF065 54.25 ZF135 61.14 ZF205 58.51 ZF066 54.95 ZF136 47.14 ZF206 77.67 ZF067 69.99 ZF137 101.88 ZF207 72.94 ZF068 54.64 ZF138 99.22 ZF208 55.34 ZF069 55.99 ZF139 69.52 ZF209 64.42 ZF070 61.26 ZF140 48.41

Several ZF-off constructs were shown to be more effective at silencing PCSK9 than WTCas9 and CRISPR-off in combination with gRNA003. The target sites and ZF sequences (F1 through F6) of the ZFP domains in these ZF-off constructs are as shown in Table 1.

Example 8: Full Specificity Screen of Constructs in Primary Human Hepatocytes

The specificity of CRISPR-off and ZF-off constructs for silencing PCSK9 is tested in primary human hepatocytes. The readouts to assess specificity are RNAseq, methylation array and whole genome bisulfite sequencing assays. Genome-wide expression and methylation changes after epigenetic editing compared to negative controls will be profiled.

Example 9: CpG Methylation Patterns

The CpG methylation patterns in human hepatocytes (e.g., primary cells or cell lines) treated with CRISPR-off or ZF-off are investigated. Hybrid capture assay is performed on bisulfite treated DNA to investigate methylation patterns at CpG sites that are induced by CRISPR-Off or ZF-Off at the 1 kb region around the PCSK9 TSS.

Example 10: Stable PCSK9 Silencing Via Epigenetic Editing in Mice with Wildtype PCSK9

The ability of CRISPR-off and ZF-off constructs to mediate epigenetic silencing of endogenous PCSK9 in vivo is tested. Constructs are delivered using a single IV administration of mRNA (and, for CRISPR-off silencing, gRNA) formulated into an LNP. Silencing is tested in wildtype mice over a period of two to six months. The readout is serum PCSK9 levels and serum cholesterol levels. A subset of each cohort is selected for liver hematoxylin and eosin (H&E) stain RNAseq and analysis. For several constructs, robust, stable, and inheritable PCSK9 silencing is observed.

Example 11: Stable PCSK9 Silencing Via Epigenetic Editing in Mice Expressing Transgenic Human PCSK9

Three different mouse strains expressing transgenic human PCSK9 are used: hPCSK9-Tg (mPCSK9+/−) heterozygous mouse, hPCSK9-Tg (mPCSK9+/+) homozygous mouse, and hPCSK9-Tg (mPCSK9−/−) mouse. The hPCSK9-Tg (mPCSK9−/−) mouse line used is C57BL/6J-Pcsk9−/− Tg (RP11-55M23-AbsI), which expresses human PCSK9 under the control of its own promoter (FIG. 10). See, e.g., Weider et al., J Biol Chem (2016) 291(32):16659-71.

The CRISPR-off and ZF-off constructs are tested. Constructs are delivered via single IV administration of mRNA/gRNA formulated into LNP. The readouts are liver H&E stain, RNAseq to measure PCSK9 mRNA levels, and AST/ASL measurements. Efficacy is also tested, including durability of PCSK9 silencing over three to four months as measured by the level of serum PCSK9 protein. A durable and significant reduction in the levels of serum PCSK9 is observed for some constructs.

Durability is tested over six to twelve months. Readouts are serum PCSK9 levels and serum cholesterol levels. A subset of the cohort is selected for liver H&E and RNAseq analysis.

Example 12: Fusion Proteins with Variant NLS Configurations

Several improved fusion protein constructs were developed using variant nuclear localization sequence (NLS) configurations to have significantly higher epi-silencing activity.

Several constructs with variant configurations of NLS domains (FIGS. 11A and 11B) were constructed and tested in PCSK9 loci in HeLa cells (FIGS. 12A-12B). The constructs were additionally tested in PCSK9 loci in Hepa1-6 (FIG. 13) and in HuH7 (FIGS. 14A-14C and FIG. 15). Exemplary fusion protein construct amino acid and DNA sequences are shown below:

Fusion Protein 1 MPKKKRKVPKKKRKVNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVD Amino Acid RYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPAR Sequence KGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDA NLS-3A-3L-dCas9- KEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGK KRAB-NLS DQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLK (SEQ ID NO: EYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHNPLEMFETVPVWRRQPVRVLSLFEDIKKE 1498) LTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFH RLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSN IPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGG PSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLI EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSI DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQSITGLYETRIDLSQLGGDSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTA QQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPPKKKRKVPKKKRKV Fusion Protein 1 ATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTCAACCATGATCAAGAATT DNA Sequence CGACCCACCTAAAGTCTACCCACCTGTGCCCGCCGAAAAAAGGAAACCCATAAGGGTGCTGT (SEQ ID NO: CACTCTTTGATGGCATCGCCACTGGTCTCCTGGTTCTTAAGGATCTGGGAATTCAGGTCGAT 1499) CGGTACATTGCTAGCGAGGTTTGTGAGGATAGTATTACAGTGGGTATGGTGCGCCACCAGGG AAAGATCATGTATGTTGGTGACGTTAGGAGCGTCACCCAGAAACATATCCAGGAGTGGGGAC CCTTTGATTTGGTGATCGGAGGTAGTCCCTGCAATGACCTTTCCATCGTGAATCCAGCCAGG AAAGGGCTGTATGAAGGGACTGGTAGGCTCTTTTTCGAGTTTTATCGCCTGCTTCACGACGC TAGACCTAAGGAAGGTGACGATAGGCCTTTCTTTTGGCTTTTTGAGAACGTCGTGGCAATGG GAGTCTCCGACAAAAGGGACATTTCTCGCTTTCTGGAATCTAACCCCGTTATGATCGATGCC AAGGAAGTTTCTGCCGCTCACAGGGCAAGGTACTTCTGGGGCAATCTGCCCGGAATGAATCG CCCACTGGCCAGTACCGTGAATGACAAACTGGAGCTGCAGGAGTGCCTGGAGCACGGAAGAA TCGCAAAGTTTTCTAAAGTCAGGACCATTACCACTCGCAGTAACTCCATAAAACAGGGTAAG GACCAGCATTTTCCCGTCTTCATGAATGAAAAGGAAGATATTCTGTGGTGCACTGAAATGGA GAGAGTTTTCGGGTTTCCCGTGCACTATACCGATGTTTCCAACATGTCCCGCCTTGCAAGAC AAAGGCTTTTGGGCCGCTCTTGGTCTGTGCCAGTGATCCGGCACTTGTTTGCTCCCCTCAAA GAGTACTTCGCTTGCGTCAGTTCCGGAAATTCAAACGCTAACTCTCGGGGTCCATCTTTCTC CAGTGGTCTCGTGCCACTGTCTCTCCGGGGCTCTCACAATCCCCTGGAGATGTTTGAGACAG TGCCAGTCTGGCGGAGGCAGCCCGTTCGCGTTCTCTCTCTGTTCGAAGATATTAAAAAGGAA CTCACCTCCCTTGGGTTCCTGGAGAGCGGGAGCGACCCCGGACAGCTTAAGCACGTGGTCGA CGTGACTGACACCGTCCGCAAAGACGTGGAGGAATGGGGCCCCTTCGATCTGGTCTATGGGG CAACCCCTCCCCTTGGGCATACATGTGATCGGCCTCCATCCTGGTACCTGTTCCAGTTTCAC AGACTCCTGCAGTATGCCAGGCCAAAGCCAGGGAGCCCAAGGCCCTTTTTCTGGATGTTCGT CGACAACCTGGTCCTGAACAAAGAAGATCTCGACGTTGCTAGTCGCTTTCTCGAAATGGAGC CCGTGACCATTCCCGACGTGCATGGCGGTTCCCTCCAGAATGCAGTCAGGGTTTGGAGCAAT ATCCCTGCCATCAGGTCAAGGCACTGGGCACTGGTTTCAGAGGAAGAGCTGTCCCTCCTTGC CCAGAACAAGCAGTCATCCAAACTGGCAGCCAAGTGGCCAACTAAGCTGGTCAAGAACTGCT TTCTTCCCCTCAGAGAATATTTTAAGTATTTCAGTACTGAACTGACTAGCAGTCTGGGAGGG CCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCAACATCTACTGA AGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAGAACCATCTG AAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGCACGTCAACC GAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAGATGGACAAGAAGTA CAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACA AGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAAC CTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCGGCTGAAGAGAAC CGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCA ACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAA GAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCA CGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCG ACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATC GAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC CTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCC TGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG AAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAA GAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACG ACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAG AACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGC CCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGA AAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAG AACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACC TGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAG CTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAA GATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACA GCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAA GTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAA CCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACA ACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGC GAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCA GCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGG AAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAG GACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACT GTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACA AAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTG ATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACA TCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCC GGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAA AGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCA CCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAG CTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCT GTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACC GGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATC GATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGA AGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGCTGATTACCC AGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCC GGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCT GGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGGAAGTGAAAGTGA TCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGC GAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCT GATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACG TGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTC TACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAA GCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGGGCCGGGACT TTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTG CAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGC CAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATT CTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAG CTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGA AGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGT TCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAAC GAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCT GAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAACACTACC TGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAAT CTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGAGCAGGCCGA GAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTG ACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATC CACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAG CGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAAGCACCGGTCGGACACTGG TGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCT CAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCCTTGGGTTA TCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCCCCAAAAAAGA AGAGAAAGGTACCGAAGAAAAAAAGAAAGGTC Fusion Protein 2 MNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGIQVDRYIASEVCEDSITV Amino Acid GMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEF Sequence YRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWG 3A-3L-NLS-dCas9- NLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEKEDI NLS-KRAB LWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNAN (SEQ ID NO: SRGPSFSSGLVPLSLRGSHNPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPG 1500) QLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPR PFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALVSE EELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGGPSSGAPPPSGGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEPKKKRKVMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHER HPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD NSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYI DGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRR QEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEE NEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILK EHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTR SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNF FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSA YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGL YETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTA QQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP Fusion Protein 2 ATGAACCACGACCAGGAATTTGACCCTCCAAAGGTTTACCCACCTGTCCCAGCTGAGAAGAG DNA Sequence GAAGCCCATCCGGGTGCTGTCTCTCTTTGATGGAATCGCTACAGGGCTCCTGGTGCTGAAGG (SEQ ID NO: ACTTGGGCATTCAGGTGGACCGCTACATTGCCTCGGAGGTGTGTGAGGACTCCATCACGGTG 1501) GGCATGGTGCGGCACCAGGGGAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAA GCATATCCAGGAGTGGGGCCCATTCGATCTGGTGATTGGGGGCAGTCCCTGCAATGACCTCT CCATCGTCAACCCTGCTCGCAAGGGCCTCTACGAGGGCACTGGCCGGCTCTTCTTTGAGTTC TACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTT TGAGAATGTGGTGGCCATGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTCGAGTCCA ACCCTGTGATGATTGATGCCAAAGAAGTGTCAGCTGCACACAGGGCCCGCTACTTCTGGGGT AACCTTCCCGGTATGAACAGGCCGTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAGGA GTGTCTGGAGCATGGCAGGATAGCCAAGTTCAGCAAAGTGAGGACCATTACTACGAGGTCAA ACTCCATAAAGCAGGGCAAAGACCAGCATTTTCCTGTCTTCATGAATGAGAAAGAGGACATC TTATGGTGCACTGAAATGGAAAGGGTATTTGGTTTCCCAGTCCACTATACTGACGTCTCCAA CATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGGTCATGGAGCGTGCCAGTCATCCGCC ACCTCTTCGCTCCGCTGAAGGAGTATTTTGCGTGTGTGTCTAGCGGCAATAGTAACGCTAAC AGCCGCGGGCCGAGCTTCAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCATAATCC CCTTGAGATGTTCGAAACCGTGCCTGTGTGGAGGAGACAGCCAGTCCGGGTGCTGTCCCTTT TTGAAGACATCAAGAAAGAGCTGACGAGTTTGGGCTTTTTGGAAAGTGGTTCTGACCCGGGA CAACTGAAGCATGTGGTTGATGTCACAGACACAGTGAGGAAGGATGTGGAGGAGTGGGGACC CTTCGATCTTGTGTACGGCGCCACACCTCCCCTGGGCCACACCTGTGACCGTCCTCCCAGCT GGTACCTGTTCCAGTTCCACCGGCTCCTGCAGTACGCACGGCCCAAGCCAGGCAGCCCCAGG CCCTTCTTCTGGATGTTCGTGGACAATCTGGTGCTGAACAAGGAAGACCTGGACGTCGCATC TCGCTTCCTGGAGATGGAGCCAGTCACCATCCCAGATGTCCACGGCGGATCCTTGCAGAATG CTGTCCGCGTGTGGAGCAACATCCCAGCCATAAGGAGCAGGCACTGGGCTCTGGTTTCGGAA GAAGAATTGTCCCTGCTGGCCCAGAACAAGCAGAGCTCGAAGCTCGCGGCCAAGTGGCCCAC CAAGCTGGTGAAGAACTGCTTTCTCCCCCTAAGAGAATATTTCAAGTATTTTTCAACAGAAC TCACTTCCTCTTTAGGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCC GGGTCCCCAACATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGG TACCTCCACAGAACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCA CCGAAGAAGGCACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCA TCTGAGCCAAAAAAGAAGAGAAAGGTAATGGACAAGAAGTACAGCATCGGCCTGGCCATCGG CACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCA AGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTC GACAGCGGAGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAG ACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACG ACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGG CACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTA CCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGG CCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGAC AACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGA AAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGA GCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGC AACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGA GGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCC AGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTG CTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGAT CAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGC TGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATC GATGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGA CGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCT TCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGG CAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTT CCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCA GAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAGC GCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCT GCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAAGTGAAAT ACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAAGCCATCGTG GACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAA GAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCC TGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAA AACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGAT CGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGC GGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAG CAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCAT GCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCG GCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAG GGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCC CGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACA GCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAA GAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAA TGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGG ACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGATAACAAAGTGCTGACTCGG AGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAA GAACTACTGGCGCCAGCTGCTGAATGCCAAGCTGATTACCCAGAGGAAGTTCGACAATCTGA CCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTG GTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAA GTACGACGAGAACGACAAACTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGG TGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCAC GCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCT GGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGA GCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTT TTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAA CGGCGAAACAGGCGAGATCGTGTGGGATAAGGGCCGGGACTTTGCCACCGTGCGGAAAGTGC TGTCTATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAA GAGTCTATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCC TAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAG TGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATG GAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGT GAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGA AGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAA TATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAA TGAGCAGAAACAGCTGTTTGTGGAACAGCACAAACACTACCTGGACGAGATCATCGAGCAGA TCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAGGTGCTGAGCGCC TACAACAAGCACAGAGACAAGCCTATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTAC CCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGA GGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTG TACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACCCAAAAAAGAAGAGAAAGGTAAG CGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAAGCACCGGTCGGACACTGG TGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCT CAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCCTTGGGTTA TCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCC Fusion Protein 3 MNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGIQVDRYIASEVCEDSITV Amino Acid GMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEF Sequence YRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWG 3A-ADD-3L-NLS- NLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEKEDI dCas9-NLS-KRAB LWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNAN (SEQ ID NO: SRGPSFSSGLVPLSLRGSHMEVKVNRRSIEDICLCCGTLQVYTRHPLFEGGLCAPCKDKFLE 1502) SLFLYDDDGHQSYCTICCSGGTLFICESPDCTRCYCFECVDILVGPGTSERINAMACWVCFL CLPFSRSGLLQRRKRWRHQLKAFHDQEGAGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSL GFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRI LQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIP GLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSS GAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEPKKKRKVMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLK DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSE LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLK SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDE TREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP Fusion Protein 3 ATGAACCATGACCAGGAATTTGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGAG DNA Sequence GAAGCCCATCCGCGTGCTGTCTCTCTTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGG (SEQ ID NO: ACCTGGGCATCCAAGTGGACCGCTACATTGCCTCCGAGGTGTGTGAGGACTCCATCACGGTG 1503) GGCATGGTGCGGCACCAGGGAAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAA GCATATCCAGGAGTGGGGCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAATGACCTCT CCATTGTCAACCCTGCCCGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTC TACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTT TGAGAATGTGGTGGCCATGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTA ACCCCGTGATGATTGACGCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGT AACCTTCCTGGCATGAACAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGA GTGTCTGGAGCACGGCAGAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGTCAA ACTCTATAAAGCAGGGCAAAGACCAGCATTTCCCCGTCTTCATGAACGAGAAGGAGGACATC CTGTGGTGCACTGAAATGGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTCTCCAA CATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGATCGTGGAGCGTGCCGGTCATCCGCC ACCTCTTCGCTCCGCTGAAGGAATATTTTGCTTGTGTGTCTAGCGGCAATAGTAACGCTAAC AGCCGCGGGCCGAGCTTCAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCATATGGA AGTCAAAGTGAACCGACGGAGCATTGAAGACATCTGCCTCTGCTGTGGAACTCTCCAGGTGT ACACTCGGCACCCCTTGTTTGAGGGAGGGTTATGTGCCCCATGTAAGGATAAGTTCCTGGAG TCCCTCTTCCTGTATGATGATGATGGACACCAGAGTTACTGCACCATCTGCTGTTCCGGGGG TACCCTGTTCATCTGTGAGAGCCCCGACTGTACCAGATGCTACTGTTTCGAGTGTGTGGACA TCCTGGTGGGCCCCGGGACCTCAGAGAGGATCAATGCCATGGCCTGCTGGGTTTGCTTCCTG TGCCTGCCCTTCTCACGGAGTGGACTGCTGCAGAGGCGCAAGAGGTGGCGGCACCAGCTGAA GGCCTTCCATGATCAAGAGGGAGCGGGCCCTATGGAGATATACAAGACAGTGTCTGCATGGA AGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGAAACATCGACAAGGTACTAAAGAGTTTG GGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAAGTACGTGGAAGATGTCAC AAATGTCGTGAGGAGAGACGTGGAGAAATGGGGCCCCTTTGACCTGGTGTACGGCTCGACGC AGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATGTTCCAGTTCCACCGGATC CTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGCGGCCCTTCTTCTGGATATTCATGGACAA TCTGCTGCTGACTGAGGATGACCAAGAGACAACTACCCGCTTCCTTCAGACAGAGGCTGTGA CCCTCCAGGATGTCCGTGGCAGAGACTACCAGAATGCTATGCGGGTGTGGAGCAACATTCCA GGGCTGAAGAGCAAGCATGCGCCCCTGACCCCAAAGGAAGAAGAGTATCTGCAAGCCCAAGT CAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGGTGAAGAACTGCCTTCTCC CGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCTCTTGGAGGGCCGAGCTCT GGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCAACATCTACTGAAGAAGGCAC CAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAGAACCATCTGAAGGTAGTG CGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGCACGTCAACCGAACCAAGT GAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAGCCAAAAAAGAAGAGAAAGGTAAT GGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCA CCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGC ATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCG GCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAG AGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCC TTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGA GGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCA CCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGC CACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCA GCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACG CCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAG CTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGAC CCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACA CCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGA GATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACC TGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTC GACCAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTA CAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGA ACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATC CACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGA CAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGG CCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGG AACTTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAA CTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACT TCACCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCC TTCCTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGT GACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAA TCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATT ATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCT GACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACC TGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTG AGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCT GAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCT TTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATT GCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGA CGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAG AGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAG GGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCA GAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAAC TGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAG GACGACTCCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAA CGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCA AGCTGATTACCCAGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAA CTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGT GGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGG AAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTT TACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGT GGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACA AGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCC AAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGG CGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATA AGGGCCGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAA AAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGA CAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCA CCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAG AGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCAT CGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTA AGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTG CAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCA CTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGC ACAAACACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTG GCCGACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAG AGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCT TCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGAC GCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCT GGGAGGCGACCCAAAAAAGAAGAGAAAGGTAAGCGGAAGTGAGACCCCAGGTACATCCGAAT CAGCAACGCCTGAAAGCACCGGTCGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTC ACCAGGGAGGAGTGGAAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCT GGAGAACTATAAGAACCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCC GGTTGGAGAAGGGAGAAGAGCCC Fusion Protein 4 MNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITV Amino Acid GMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEF Sequence YRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWG 3A-ADD-h3L-NLS- NLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDI dCas 9-NLS-KRAB LWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNAN (SEQ ID NO: SRGPSFSSGLVPLSLRGSHMEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKELD 1504) ALFLYDDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYL CLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSL GFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQ YARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAI RSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGGPSSG APPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEPKKKRKVMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGH FLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQL PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFL AAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLLKII KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIA NLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG IKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSEL DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDERKDFQFY KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILA DANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDA TLIHQSITGLYETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFT REEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP Fusion Protein 4 ATGAACCATGATCAAGAATTCGACCCACCTAAAGTCTACCCACCTGTGCCCGCCGAAAAAAG DNA Sequence GAAACCCATAAGGGTGCTGTCACTCTTTGATGGCATCGCCACTGGTCTCCTGGTTCTTAAGG (SEQ ID NO: ATCTGGGAATTCAGGTCGATCGGTACATTGCTAGCGAGGTTTGTGAGGATAGTATTACAGTG 1505) GGTATGGTGCGCCACCAGGGAAAGATCATGTATGTTGGTGACGTTAGGAGCGTCACCCAGAA ACATATCCAGGAGTGGGGACCCTTTGATTTGGTGATCGGAGGTAGTCCCTGCAATGACCTTT CCATCGTGAATCCAGCCAGGAAAGGGCTGTATGAAGGGACTGGTAGGCTCTTTTTCGAGTTT TATCGCCTGCTTCACGACGCTAGACCTAAGGAAGGTGACGATAGGCCTTTCTTTTGGCTTTT TGAGAACGTCGTGGCAATGGGAGTCTCCGACAAAAGGGACATTTCTCGCTTTCTGGAATCTA ACCCCGTTATGATCGATGCCAAGGAAGTTTCTGCCGCTCACAGGGCAAGGTACTTCTGGGGC AATCTGCCCGGAATGAATCGCCCACTGGCCAGTACCGTGAATGACAAACTGGAGCTGCAGGA GTGCCTGGAGCACGGAAGAATCGCAAAGTTTTCTAAAGTCAGGACCATTACCACTCGCAGTA ACTCCATAAAACAGGGTAAGGACCAGCATTTTCCCGTCTTCATGAATGAAAAGGAAGATATT CTGTGGTGCACTGAAATGGAGAGAGTTTTCGGGTTTCCCGTGCACTATACCGATGTTTCCAA CATGTCCCGCCTTGCAAGACAAAGGCTTTTGGGCCGCTCTTGGTCTGTGCCAGTGATCCGGC ACTTGTTTGCTCCCCTCAAAGAGTACTTCGCTTGCGTCAGTTCCGGAAATTCAAACGCTAAC TCTCGGGGTCCATCTTTCTCCAGTGGTCTCGTGCCACTGTCTCTCCGGGGCTCTCACATGGA AGTCAAGGCTAACCAGCGAAATATAGAAGACATCTGCATCTGCTGCGGAAGTCTCCAGGTTC ACACACAGCACCCTCTGTTTGAGGGAGGGATCTGCGCCCCATGTAAGGACAAGTTCCTGGAT GCCCTCTTCCTGTACGACGATGACGGGTACCAATCCTACTGCTCCATCTGCTGCTCCGGAGA GACGCTGCTCATCTGCGGAAACCCTGATTGCACCCGATGCTACTGCTTCGAGTGTGTGGATA GCCTGGTCGGCCCCGGGACCTCGGGGAAGGTGCACGCCATGAGCAACTGGGTGTGCTACCTG TGCCTGCCGTCCTCCCGAAGCGGGCTGCTGCAGCGTCGGAGGAAGTGGCGCAGCCAGCTCAA GGCCTTCTACGACCGAGAGTCGGAGAATCCCCTGGAGATGTTTGAGACAGTGCCAGTCTGGC GGAGGCAGCCCGTTCGCGTTCTCTCTCTGTTCGAAGATATTAAAAAGGAACTCACCTCCCTT GGGTTCCTGGAGAGCGGGAGCGACCCCGGACAGCTTAAGCACGTGGTCGACGTGACTGACAC CGTCCGCAAAGACGTGGAGGAATGGGGCCCCTTCGATCTGGTCTATGGGGCAACCCCTCCCC TTGGGCATACATGTGATCGGCCTCCATCCTGGTACCTGTTCCAGTTTCACAGACTCCTGCAG TATGCCAGGCCAAAGCCAGGGAGCCCAAGGCCCTTTTTCTGGATGTTCGTCGACAACCTGGT CCTGAACAAAGAAGATCTCGACGTTGCTAGTCGCTTTCTCGAAATGGAGCCCGTGACCATTC CCGACGTGCATGGCGGTTCCCTCCAGAATGCAGTCAGGGTTTGGAGCAATATCCCTGCCATC AGGTCAAGGCACTGGGCACTGGTTTCAGAGGAAGAGCTGTCCCTCCTTGCCCAGAACAAGCA GTCATCCAAACTGGCAGCCAAGTGGCCAACTAAGCTGGTCAAGAACTGCTTTCTTCCCCTCA GAGAATATTTTAAGTATTTCAGTACTGAACTGACTAGCAGTCTGGGAGGGCCGAGCTCTGGC GCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCAACATCTACTGAAGAAGGCACCAG CGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAGAACCATCTGAAGGTAGTGCGC CTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGCACGTCAACCGAACCAAGTGAA GGATCTGCCCCTGGGACCAGCACTGAACCATCTGAGCCAAAAAAGAAGAGAAAGGTAATGGA CAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCG ACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATC AAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCGGCT GAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGA TCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTC CTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCG ACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCAC TTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCT GGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCA AGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTG CCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCC CAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCT ACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTG GCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGA CCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGAC CAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAA GTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACA GAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCAC CTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAA CCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCA GGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC TTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTT CGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA CCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC CTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGAC CGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCT CCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGAC CCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGT TCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGC CGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAA GTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTA AAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCC AATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGA GCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGA ACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGC ATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAA CGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGG ACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGAC GACTCCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGT GCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGC TGATTACCCAGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTG GATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGC ACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGGAAG TGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTAC AAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGG AACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGG TGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAG TACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGA GATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGG GCCGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAG ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAA GCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGT GTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGA CTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGT ACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACA AACACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCC GACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGA GCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCA AGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCC ACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGG AGGCGACCCAAAAAAGAAGAGAAAGGTAAGCGGAAGTGAGACCCCAGGTACATCCGAATCAG CAACGCCTGAAAGCACCGGTCGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACC AGGGAGGAGTGGAAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGA GAACTATAAGAACCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGT TGGAGAAGGGAGAAGAGCCC Fusion Protein 5 MPKKKRKVPKKKRKVNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGIQVD Amino Acid RYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPAR Sequence KGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDA NLS-3A-3L-dCas9- KEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGK KRAB-NLS DQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLK (SEQ ID NO: EYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHNPLEMFETVPVWRRQPVRVLSLFEDIKKE 1506) LTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFH RLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSN IPAIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGG PSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLI EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAK NLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK DELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKE LGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSI DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELDKA GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFF YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI HQSITGLYETRIDLSQLGGDSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTA QQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKKRK V Fusion Protein 5 ATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTCAACCACGACCAGGAATT DNA Sequence CGACCCTCCAAAGGTTTACCCACCTGTCCCAGCTGAGAAGAGGAAGCCCATCCGGGTGCTGT (SEQ ID NO: CTCTCTTTGATGGAATCGCTACAGGGCTCCTGGTGCTGAAGGACTTGGGCATTCAGGTGGAC 1507) CGCTACATTGCCTCGGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCAGGG GAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGGGCC CATTCGATCTGGTGATTGGGGGCAGTCCCTGCAATGACCTCTCCATCGTCAACCCTGCTCGC AAGGGCCTCTACGAGGGCACTGGCCGGCTCTTCTTTGAGTTCTACCGCCTCCTGCATGATGC GCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCATGG GCGTTAGTGACAAGAGGGACATCTCGCGATTTCTCGAGTCCAACCCTGTGATGATTGATGCC AAAGAAGTGTCAGCTGCACACAGGGCCCGCTACTTCTGGGGTAACCTTCCCGGTATGAACAG GCCGTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAGGAGTGTCTGGAGCATGGCAGGA TAGCCAAGTTCAGCAAAGTGAGGACCATTACTACGAGGTCAAACTCCATAAAGCAGGGCAAA GACCAGCATTTTCCTGTCTTCATGAATGAGAAAGAGGACATCTTATGGTGCACTGAAATGGA AAGGGTATTTGGTTTCCCAGTCCACTATACTGACGTCTCCAACATGAGCCGCTTGGCGAGGC AGAGACTGCTGGGCCGGTCATGGAGCGTGCCAGTCATCCGCCACCTCTTCGCTCCGCTGAAG GAGTATTTTGCGTGTGTGTCTAGCGGCAATAGTAACGCTAACAGCCGCGGGCCGAGCTTCAG CAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCATAATCCCCTTGAGATGTTCGAAACCG TGCCTGTGTGGAGGAGACAGCCAGTCCGGGTGCTGTCCCTTTTTGAAGACATCAAGAAAGAG CTGACGAGTTTGGGCTTTTTGGAAAGTGGTTCTGACCCGGGACAACTGAAGCATGTGGTTGA TGTCACAGACACAGTGAGGAAGGATGTGGAGGAGTGGGGACCCTTCGATCTTGTGTACGGCG CCACACCTCCCCTGGGCCACACCTGTGACCGTCCTCCCAGCTGGTACCTGTTCCAGTTCCAC CGGCTCCTGCAGTACGCACGGCCCAAGCCAGGCAGCCCCAGGCCCTTCTTCTGGATGTTCGT GGACAATCTGGTGCTGAACAAGGAAGACCTGGACGTCGCATCTCGCTTCCTGGAGATGGAGC CAGTCACCATCCCAGATGTCCACGGCGGATCCTTGCAGAATGCTGTCCGCGTGTGGAGCAAC ATCCCAGCCATAAGGAGCAGGCACTGGGCTCTGGTTTCGGAAGAAGAATTGTCCCTGCTGGC CCAGAACAAGCAGAGCTCGAAGCTCGCGGCCAAGTGGCCCACCAAGCTGGTGAAGAACTGCT TTCTCCCCCTAAGAGAATATTTCAAGTATTTTTCAACAGAACTCACTTCCTCTTTAGGAGGG CCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCAACATCTACTGA AGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAGAACCATCTG AAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGCACGTCAACC GAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAGATGGACAAGAAGTA CAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACA AGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAAC CTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCGGCTGAAGAGAAC CGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCA ACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAA GAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCA CGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCG ACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATC GAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGAC CTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCC TGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG AAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAA GAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACG ACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAG AACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGC CCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGA AAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAG AACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACC TGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAG CTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAA GATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACA GCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAA GTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAA CCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACA ACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGC GAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCA GCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGG AAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAG GACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACT GTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACA AAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTG ATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGG CTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACA TCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCC GGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAA AGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCA CCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAG CTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCT GTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACC GGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATC GATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGA AGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGCTGATTACCC AGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCC GGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCT GGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGGAAGTGAAAGTGA TCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGC GAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCT GATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACG TGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTC TACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAA GCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGGGCCGGGACT TTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTG CAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGC CAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATT CTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAG CTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGA AGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGT TCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAAC GAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCT GAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAACACTACC TGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAAT CTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGAGCAGGCCGA GAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTG ACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATC CACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAG CGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAAGCACCGGTCGGACACTGG TGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCT CAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCCTTGGGTTA TCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCCAGCGCTGATT ACAAAGATGATGACGATAAAGCCCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAG GTC Fusion Protein 6 MPKKKRKVPKKKRKVNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVD Amino Acid RYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPAR Sequence KGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDA NLS-3A-3L-dCas9- KEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGK KRAB-NLS DQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLK (SEQ ID NO: EYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLERNIDK 1508) VLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMF QFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRV WSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGI KELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQK GNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLD TAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKK RKV Fusion Protein 6 ATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAACCATGACCAGGA DNA Sequence ATTCGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGAGGAAGCCCATCCGCGTGC (SEQ ID NO: TGTCTCTCTTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGGACCTGGGCATCCAAGTG 1509) GACCGCTACATTGCCTCCGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCA GGGAAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGG GCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAATGACCTCTCCATTGTCAACCCTGCC CGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCTCCTGCATGA TGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCA TGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTAACCCCGTGATGATTGAC GCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGTAACCTTCCTGGCATGAA CAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGAGTGTCTGGAGCACGGCA GAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGTCAAACTCTATAAAGCAGGGC AAAGACCAGCATTTCCCCGTCTTCATGAACGAGAAGGAGGACATCCTGTGGTGCACTGAAAT GGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTCTCCAACATGAGCCGCTTGGCGA GGCAGAGACTGCTGGGCCGATCGTGGAGCGTGCCGGTCATCCGCCACCTCTTCGCTCCGCTG AAGGAATATTTTGCTTGTGTGTCTAGCGGCAATAGTAACGCTAACAGCCGCGGGCCGAGCTT CAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCATATGGGCCCTATGGAGATATACA AGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGAAACATCGAC AAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAA GTACGTGGAAGATGTCACAAATGTCGTGAGGAGAGACGTGGAGAAATGGGGCCCCTTTGACC TGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATG TTCCAGTTCCACCGGATCCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGCGGCCCTTCTT CTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACAACTACCCGCTTCC TTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGAGACTACCAGAATGCTATGCGG GTGTGGAGCAACATTCCAGGGCTGAAGAGCAAGCATGCGCCCCTGACCCCAAAGGAAGAAGA GTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGG TGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCT CTTGGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCAAC ATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAG AACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGC ACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAGATGGA CAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCG ACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATC AAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCGGCT GAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGA TCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTC CTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCG ACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCAC TTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCT GGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCA AGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTG CCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCC CAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCT ACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTG GCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGA CCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGAC CAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAA GTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACA GAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCAC CTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAA CCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCA GGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC TTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTT CGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA CCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC CTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGAC CGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCT CCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGAC CCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGT TCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGC CGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAA GTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTA AAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCC AATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGA GCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGA ACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGC ATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAA CGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGG ACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGAC GACTCCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGT GCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGC TGATTACCCAGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTG GATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGC ACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGGAAG TGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTAC AAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGG AACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGG TGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAG TACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGA GATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGG GCCGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAG ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAA GCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGT GTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGA CTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGT ACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACA AACACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCC GACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGA GCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCA AGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCC ACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGG AGGCGACAGCGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAAGCACCGGTC GGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTG GACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTC CTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCCA GCGCTGATTACAAAGATGATGACGATAAAGCCCCAAAAAAGAAGAGAAAGGTACCGAAGAAA AAAAGAAAGGTC Fusion Protein 7 MPKKKRKVPKKKRKVNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVD Amino Acid RYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPAR Sequence KGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDA NLS-3A-3L-dCas9- KEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGK ZIM-NLS DQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLK (SEQ ID NO: EYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDK 1510) VLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMF QFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRV WSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHF LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNEDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGI KELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQK GNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSGSETPGTSESATPESTGMNNSQGRVTFEDVTVNFTQGEW QRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNG DIGGQIWKPKDVKESLSADYKDDDDKAPKKKRKVPKKKRKV Fusion Protein 7 ATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAACCATGACCAGGA DNA Sequence ATTCGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGAGGAAGCCCATCCGCGTGC (SEQ ID NO: TGTCTCTCTTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGGACCTGGGCATCCAAGTG 1511) GACCGCTACATTGCCTCCGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCA GGGAAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGG GCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAATGACCTCTCCATTGTCAACCCTGCC CGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCTCCTGCATGA TGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCA TGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTAACCCCGTGATGATTGAC GCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGTAACCTTCCTGGCATGAA CAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGAGTGTCTGGAGCACGGCA GAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGTCAAACTCTATAAAGCAGGGC AAAGACCAGCATTTCCCCGTCTTCATGAACGAGAAGGAGGACATCCTGTGGTGCACTGAAAT GGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTCTCCAACATGAGCCGCTTGGCGA GGCAGAGACTGCTGGGCCGATCGTGGAGCGTGCCGGTCATCCGCCACCTCTTCGCTCCGCTG AAGGAATATTTTGCTTGTGTGTCTAGCGGCAATAGTAACGCTAACAGCCGCGGGCCGAGCTT CAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCATATGGGCCCTATGGAGATATACA AGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGAAACATCGAC AAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAA GTACGTGGAAGATGTCACAAATGTCGTGAGGAGAGACGTGGAGAAATGGGGCCCCTTTGACC TGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATG TTCCAGTTCCACCGGATCCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGCGGCCCTTCTT CTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACAACTACCCGCTTCC TTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGAGACTACCAGAATGCTATGCGG GTGTGGAGCAACATTCCAGGGCTGAAGAGCAAGCATGCGCCCCTGACCCCAAAGGAAGAAGA GTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGG TGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCT CTTGGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCAAC ATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAG AACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGC ACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAGATGGA CAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCG ACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATC AAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCGGCT GAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGA TCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTC CTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCG ACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCAC TTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCT GGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCA AGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTG CCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCC CAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCT ACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTG GCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGA CCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGAC CAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAA GTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACA GAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCAC CTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAA CCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCA GGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC TTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTT CGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA CCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC CTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGAC CGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCT CCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGAC CCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGT TCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGC CGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAA GTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTA AAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCC AATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGA GCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGA ACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGC ATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAA CGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGG ACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGAC GACTCCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGT GCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGC TGATTACCCAGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTG GATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGC ACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGGAAG TGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTAC AAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGG AACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGG TGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAG TACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGA GATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGG GCCGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAG ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAA GCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGT GTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGA CTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGT ACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACA AACACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCC GACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGA GCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCA AGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCC ACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGG AGGCGACAGCGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAAGCACCGGTA TGAACAATTCACAGGGGAGAGTGACATTCGAAGACGTGACCGTGAACTTCACCCAGGGAGAA TGGCAGCGCTTGAACCCAGAACAAAGGAACCTCTATCGGGACGTGATGCTGGAAAACTACTC AAATTTGGTGAGCGTTGGGCAGGGTGAGACCACTAAGCCTGACGTGATCCTGAGATTGGAAC AGGGCAAGGAGCCTTGGCTCGAGGAAGAGGAAGTCCTGGGCTCAGGGAGGGCCGAGAAAAAC GGTGATATAGGAGGCCAGATATGGAAGCCTAAGGACGTCAAGGAGAGCCTGAGCGCTGATTA CAAAGATGATGACGATAAAGCCCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGG TC Fusion Protein 8 MPKKKRKVPKKKRKVNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGIQVD Amino Acid RYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPAR Sequence KGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDA NLS-3A-3L-dCas9- KEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGK ZFP-NLS DQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLK (SEQ ID NO: EYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLERNIDK 1512) VLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMF QFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRV WSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK KNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHE LIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK DKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGI KELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYK VREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQK GNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSGSETPGTSESATPESTGNKKLEAVGTGIEPKAMSQGLVT FGDVAVDFSQEEWEWLNPIQRNLYRKVMLENYRNLASLGLCVSKPDVISSLEQGKEPWSADY KDDDDKAPKKKRKVPKKKRKV Fusion Protein 8 ATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAACCATGACCAGGA DNA Sequence ATTCGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGAGGAAGCCCATCCGCGTGC (SEQ ID NO: TGTCTCTCTTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGGACCTGGGCATCCAAGTG 1513) GACCGCTACATTGCCTCCGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGTGCGGCACCA GGGAAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATATCCAGGAGTGGG GCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAATGACCTCTCCATTGTCAACCCTGCC CGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCTCCTGCATGA TGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATGTGGTGGCCA TGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTAACCCCGTGATGATTGAC GCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGTAACCTTCCTGGCATGAA CAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGAGTGTCTGGAGCACGGCA GAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGTCAAACTCTATAAAGCAGGGC AAAGACCAGCATTTCCCCGTCTTCATGAACGAGAAGGAGGACATCCTGTGGTGCACTGAAAT GGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTCTCCAACATGAGCCGCTTGGCGA GGCAGAGACTGCTGGGCCGATCGTGGAGCGTGCCGGTCATCCGCCACCTCTTCGCTCCGCTG AAGGAATATTTTGCTTGTGTGTCTAGCGGCAATAGTAACGCTAACAGCCGCGGGCCGAGCTT CAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCATATGGGCCCTATGGAGATATACA AGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGAAACATCGAC AAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGGAACGCTGAA GTACGTGGAAGATGTCACAAATGTCGTGAGGAGAGACGTGGAGAAATGGGGCCCCTTTGACC TGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGCTGGTACATG TTCCAGTTCCACCGGATCCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGCGGCCCTTCTT CTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACAACTACCCGCTTCC TTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGAGACTACCAGAATGCTATGCGG GTGTGGAGCAACATTCCAGGGCTGAAGAGCAAGCATGCGCCCCTGACCCCAAAGGAAGAAGA GTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTGACCTCCTGG TGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAACTCACTTCCT CTTGGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGGGTCCCCAAC ATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTACCTCCACAG AACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACCGAAGAAGGC ACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATCTGAGATGGA CAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCG ACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATC AAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCGGCT GAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGA TCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTC CTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCG ACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCAC TTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCT GGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCA AGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTG CCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCC CAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCT ACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTG GCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGA CCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGAC CAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAA GTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACA GAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCAC CTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAA CCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCA GGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC TTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTT CGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA CCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC CTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGAC CGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCT CCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGAC CCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGT TCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGC CGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAA GTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTA AAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCC AATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGA GCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGA ACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGC ATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAA CGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGG ACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGAC GACTCCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGT GCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGC TGATTACCCAGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTG GATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGC ACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGGAAG TGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTAC AAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGG AACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGG TGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAG TACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGA GATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGG GCCGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAG ACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAA GCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCG TGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGT GTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGA CTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGT ACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACA AACACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCC GACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGA GCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCA AGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCC ACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGG AGGCGACAGCGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAAGCACCGGTA ACAAAAAGCTTGAGGCCGTCGGAACCGGAATCGAACCAAAAGCAATGTCCCAGGGTTTGGTG ACATTTGGCGACGTGGCTGTCGATTTTTCCCAGGAAGAGTGGGAGTGGCTCAATCCTATCCA GAGGAACTTGTACCGGAAGGTGATGCTGGAGAATTATAGAAATTTGGCATCACTGGGGTTGT GCGTTAGCAAACCAGATGTTATATCTTCCCTGGAACAGGGAAAGGAGCCCTGGAGCGCTGAT TACAAAGATGATGACGATAAAGCCCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAA GGTC Fusion Protein 9 MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLG Variant 1 IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV Amino Acid NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV Sequence MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI NLS-NLS-3A-h3L- KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF dCas 9-KOX1KRAB- APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEPSMDVILVGSSEL NLS-NLS SSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLY (SEQ ID NO: DDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSS 1514) RSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLES GSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPK PGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHW ALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGGPSSGAPPPS GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYID GGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSESATPESRTLVTFKDVFVDFTREEWKLL DTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKK KRKV Fusion Protein 9 ATGGGTACCATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAATCA Variant 1 CGATCAGGAGTTCGACCCCCCTAAGGTGTACCCACCAGTGCCTGCAGAGAAGAGGAAGCCAA DNA Sequence TCCGGGTGCTGAGCCTGTTTGATGGCATCGCCACCGGCCTGCTGGTGCTGAAGGATCTGGGC (SEQ ID NO: ATCCAGGTGGACCGGTACATCGCCTCCGAGGTGTGCGAGGATTCTATCACCGTGGGCATGGT 1515) GCGCCACCAGGGCAAGATCATGTATGTGGGCGACGTGCGGTCCGTGACACAGAAGCACATCC AGGAGTGGGGCCCATTCGATCTGGTGATCGGCGGCAGCCCCTGTAATGACCTGTCCATCGTG AACCCTGCAAGGAAGGGACTGTACGAGGGAACCGGCCGGCTGTTCTTTGAGTTTTATAGACT GCTGCACGACGCCAGGCCTAAGGAGGGCGACGATAGACCATTCTTTTGGCTGTTCGAGAATG TGGTGGCTATGGGCGTGAGCGATAAGAGGGACATCTCCAGGTTTCTGGAGTCTAACCCCGTG ATGATCGATGCAAAGGAGGTGTCCGCCGCACACAGAGCCAGGTATTTCTGGGGCAATCTGCC AGGAATGAACAGGCCACTGGCAAGCACCGTGAATGACAAGCTGGAGCTGCAGGAGTGCCTGG AGCACGGAAGGATCGCCAAGTTTTCCAAGGTGCGCACAATCACCACACGGAGCAATTCCATC AAGCAGGGCAAGGATCAGCACTTCCCCGTGTTCATGAACGAGAAGGAGGACATCCTGTGGTG TACCGAGATGGAGAGAGTGTTCGGCTTTCCAGTGCACTACACAGACGTGTCTAACATGAGCA GGCTGGCAAGGCAGCGGCTGCTGGGCAGATCTTGGAGCGTGCCCGTGATCAGGCACCTGTTC GCCCCTCTGAAGGAGTATTTTGCCTGCGTGAGCAGCGGCAACTCCAATGCCAACAGCCGGGG CCCCTCTTTCAGCTCCGGATTGGTGCCTCTGAGCCTGAGGGGCTCCCACATGGCAGCAATCC CCGCCCTGGACCCCGAGGCCGAGCCTAGCATGGACGTGATCCTGGTGGGCTCTAGCGAGCTG TCCTCTAGCGTGTCTCCAGGAACCGGAAGGGATCTGATCGCATACGAGGTGAAGGCCAATCA GCGGAACATCGAGGACATCTGTATCTGCTGTGGCAGCCTGCAGGTGCACACACAGCACCCAC TGTTCGAGGGAGGAATCTGCGCACCCTGTAAGGATAAGTTCCTGGACGCCCTGTTTCTGTAC GACGATGACGGCTACCAGTCCTATTGCTCTATCTGCTGTTCCGGCGAGACCCTGCTGATCTG CGGCAATCCAGATTGTACAAGGTGCTATTGTTTTGAGTGCGTGGACTCTCTGGTGGGACCAG GCACCAGCGGAAAGGTGCACGCCATGTCCAACTGGGTGTGCTACCTGTGCCTGCCATCCTCT CGCAGCGGACTGCTGCAGCGGAGAAGGAAGTGGAGATCCCAGCTGAAGGCCTTCTATGATAG GGAGTCTGAGAACCCCCTGGAGATGTTTGAGACCGTGCCAGTGTGGCGCCGGCAGCCCGTGA GGGTGCTGAGCCTGTTCGAGGATATCAAGAAGGAGCTGACATCCCTGGGCTTTCTGGAGTCC GGCTCTGACCCCGGACAGCTGAAGCACGTGGTGGATGTGACCGACACAGTGCGGAAGGATGT GGAGGAGTGGGGCCCTTTCGACCTGGTGTACGGAGCAACCCCTCCACTGGGACACACATGCG ACAGACCCCCTTCTTGGTACCTGTTCCAGTTTCACCGCCTGCTGCAGTATGCAAGGCCAAAG CCAGGCAGCCCTAGACCATTCTTTTGGATGTTCGTGGATAATCTGGTGCTGAACAAGGAGGA TCTGGACGTGGCCAGCAGGTTTCTGGAGATGGAGCCAGTGACCATCCCAGACGTGCACGGCG GCTCCCTGCAGAATGCCGTGCGCGTGTGGTCTAACATCCCTGCCATCAGAAGCAGGCACTGG GCACTGGTGAGCGAGGAGGAGCTGTCCCTGCTGGCCCAGAATAAGCAGAGCAGCAAGCTGGC CGCCAAGTGGCCTACAAAGCTGGTGAAGAACTGCTTCCTGCCACTGCGGGAGTACTTCAAGT ATTTTTCCACCGAGCTGACATCTAGCCTGGGAGGACCCTCCTCTGGCGCCCCACCACCTAGC GGCGGCTCCCCTGCCGGCTCTCCAACCAGCACAGAGGAGGGCACCAGCGAGTCCGCCACACC AGAGTCTGGACCTGGCACCAGCACAGAGCCATCCGAGGGCTCTGCCCCAGGCTCTCCTGCAG GCAGCCCTACCTCCACCGAAGAGGGCACCAGCACAGAGCCTTCTGAGGGCAGCGCCCCAGGC ACCTCTACAGAGCCAAGCGAGCTCGAGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCAC CAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACG GAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACA GCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCA CCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCC TGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAA CCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCA GACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAAC CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGA TGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA TCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTG AGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGAC GGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGG CACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCG CATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCC CAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCC CAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACG TGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAA AATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGG GCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAAC GAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGA GGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGC GGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAG TCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCA GCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCC AGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGC ATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCC GCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAA CACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGG GCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACG CCATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGC GACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCA AGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTG GAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTA CGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCC CACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGA AAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTC AAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGG CGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGA GCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAG TCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAA GAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGG AAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAA AAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGA GAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATAT GTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA GCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTAC AACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCT GACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGT ACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC GAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGG AGTCGACGGATCCAGCGGCTCCGAGACCCCAGGCACATCTGAGAGCGCCACCCCTGAGTCCC GGACCCTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTG GACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTC CTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCCA GCGCTGATTACAAAGATGATGACGATAAAGCCCCAAAAAAGAAGAGAAAGGTACCGAAGAAA AAAAGAAAGGTCTGA Fusion Protein 9 MPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQV Variant 2 DRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPA Amino Acid RKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMID Sequence AKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQG NLS-NLS-3A-h3L- KDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPL dCas 9-KOX1KRAB- KEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEPSMDVILVGSSELSSS NLS-NLS VSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLYDDD (SEQ ID NO: GYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSSRSG 1523) LLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSD PGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGS PRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALV SEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSPGGPSSGAPPPSGGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLEDSGE TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIA LSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDI LRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGA SQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDF YPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKN RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDA YLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNF LYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDSPKKKRKVGVDGSSGSETPGTSESATPESRTLVTFKDVFVDFTREEWKLLDTA QQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKKRK V Fusion Protein 9 ATGGGTACCATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAATCA Variant 2 CGATCAGGAGTTCGACCCCCCTAAGGTGTACCCACCAGTGCCTGCAGAGAAGAGGAAGCCAA DNA Sequence TCCGGGTGCTGAGCCTGTTTGATGGCATCGCCACCGGCCTGCTGGTGCTGAAGGATCTGGGC (SEQ ID NO: ATCCAGGTGGACCGGTACATCGCCTCCGAGGTGTGCGAGGATTCTATCACCGTGGGCATGGT 1524) GCGCCACCAGGGCAAGATCATGTATGTGGGCGACGTGCGGTCCGTGACACAGAAGCACATCC AGGAGTGGGGCCCATTCGATCTGGTGATCGGCGGCAGCCCCTGTAATGACCTGTCCATCGTG AACCCTGCAAGGAAGGGACTGTACGAGGGAACCGGCCGGCTGTTCTTTGAGTTTTATAGACT GCTGCACGACGCCAGGCCTAAGGAGGGCGACGATAGACCATTCTTTTGGCTGTTCGAGAATG TGGTGGCTATGGGCGTGAGCGATAAGAGGGACATCTCCAGGTTTCTGGAGTCTAACCCCGTG ATGATCGATGCAAAGGAGGTGTCCGCCGCACACAGAGCCAGGTATTTCTGGGGCAATCTGCC AGGAATGAACAGGCCACTGGCAAGCACCGTGAATGACAAGCTGGAGCTGCAGGAGTGCCTGG AGCACGGAAGGATCGCCAAGTTTTCCAAGGTGCGCACAATCACCACACGGAGCAATTCCATC AAGCAGGGCAAGGATCAGCACTTCCCCGTGTTCATGAACGAGAAGGAGGACATCCTGTGGTG TACCGAGATGGAGAGAGTGTTCGGCTTTCCAGTGCACTACACAGACGTGTCTAACATGAGCA GGCTGGCAAGGCAGCGGCTGCTGGGCAGATCTTGGAGCGTGCCCGTGATCAGGCACCTGTTC GCCCCTCTGAAGGAGTATTTTGCCTGCGTGAGCAGCGGCAACTCCAATGCCAACAGCCGGGG CCCCTCTTTCAGCTCCGGATTGGTGCCTCTGAGCCTGAGGGGCTCCCACATGGCAGCAATCC CCGCCCTGGACCCCGAGGCCGAGCCTAGCATGGACGTGATCCTGGTGGGCTCTAGCGAGCTG TCCTCTAGCGTGTCTCCAGGAACCGGAAGGGATCTGATCGCATACGAGGTGAAGGCCAATCA GCGGAACATCGAGGACATCTGTATCTGCTGTGGCAGCCTGCAGGTGCACACACAGCACCCAC TGTTCGAGGGAGGAATCTGCGCACCCTGTAAGGATAAGTTCCTGGACGCCCTGTTTCTGTAC GACGATGACGGCTACCAGTCCTATTGCTCTATCTGCTGTTCCGGCGAGACCCTGCTGATCTG CGGCAATCCAGATTGTACAAGGTGCTATTGTTTTGAGTGCGTGGACTCTCTGGTGGGACCAG GCACCAGCGGAAAGGTGCACGCCATGTCCAACTGGGTGTGCTACCTGTGCCTGCCATCCTCT CGCAGCGGACTGCTGCAGCGGAGAAGGAAGTGGAGATCCCAGCTGAAGGCCTTCTATGATAG GGAGTCTGAGAACCCCCTGGAGATGTTTGAGACCGTGCCAGTGTGGCGCCGGCAGCCCGTGA GGGTGCTGAGCCTGTTCGAGGATATCAAGAAGGAGCTGACATCCCTGGGCTTTCTGGAGTCC GGCTCTGACCCCGGACAGCTGAAGCACGTGGTGGATGTGACCGACACAGTGCGGAAGGATGT GGAGGAGTGGGGCCCTTTCGACCTGGTGTACGGAGCAACCCCTCCACTGGGACACACATGCG ACAGACCCCCTTCTTGGTACCTGTTCCAGTTTCACCGCCTGCTGCAGTATGCAAGGCCAAAG CCAGGCAGCCCTAGACCATTCTTTTGGATGTTCGTGGATAATCTGGTGCTGAACAAGGAGGA TCTGGACGTGGCCAGCAGGTTTCTGGAGATGGAGCCAGTGACCATCCCAGACGTGCACGGCG GCTCCCTGCAGAATGCCGTGCGCGTGTGGTCTAACATCCCTGCCATCAGAAGCAGGCACTGG GCACTGGTGAGCGAGGAGGAGCTGTCCCTGCTGGCCCAGAATAAGCAGAGCAGCAAGCTGGC CGCCAAGTGGCCTACAAAGCTGGTGAAGAACTGCTTCCTGCCACTGCGGGAGTACTTCAAGT ATTTTTCCACCGAGCTGACATCTAGCCCCGGAGGACCCTCCTCTGGCGCCCCACCACCTAGC GGCGGCTCCCCTGCCGGCTCTCCAACCAGCACAGAGGAGGGCACCAGCGAGTCCGCCACACC AGAGTCTGGACCTGGCACCAGCACAGAGCCATCCGAGGGCTCTGCCCCAGGCTCTCCTGCAG GCAGCCCTACCTCCACCGAAGAGGGCACCAGCACAGAGCCTTCTGAGGGCAGCGCCCCAGGC ACCTCTACAGAGCCAAGCGAGCTCGAGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCAC CAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACG GAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACA GCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCA CCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCC TGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAA CCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCA GACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAAC CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGA TGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA TCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTG AGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGAC GGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGG CACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCG CATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCC CAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCC CAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACG TGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAA AATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGG GCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAAC GAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGA GGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGC GGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAG TCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCA GCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCC AGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGC ATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCC GCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAA CACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGG GCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACG CCATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGC GACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCA AGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTG GAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTA CGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCC CACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGA AAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTC AAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGG CGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGA GCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAG TCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAA GAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGG AAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAA AAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGA GAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATAT GTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA GCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTAC AACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCT GACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGT ACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC GAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGG AGTCGACGGATCCAGCGGCTCCGAGACCCCAGGCACATCTGAGAGCGCCACCCCTGAGTCCC GGACCCTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTG GACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTC CTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAGCCCA GCGCTGATTACAAAGATGATGACGATAAAGCCCCAAAAAAGAAGAGAAAGGTACCGAAGAAA AAAAGAAAGGTCTGA Fusion Protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLG 10 Amino Acid IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV Sequence NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV NLS-NLS-3A-h3L- MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI dCas9-ZFP-28- KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF NLS-NLS APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEPSMDVILVGSSEL (SEQ ID NO: SSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLY 1516) DDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSS RSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLES GSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPK PGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHW ALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGGPSSGAPPPS GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLEGN LIALSLGLTPNFKSNEDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYID GGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSESATPESTGNKKLEAVGTGIEPKAMSQG LVTFGDVAVDFSQEEWEWLNPIQRNLYRKVMLENYRNLASLGLCVSKPDVISSLEQGKEPWS ADYKDDDDKAPKKKRKVPKKKRKV Fusion Protein ATGGGTACCATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAATCA 10 DNA Sequence CGATCAGGAGTTCGACCCCCCTAAGGTGTACCCACCAGTGCCTGCAGAGAAGAGGAAGCCAA (SEQ ID NO: TCCGGGTGCTGAGCCTGTTTGATGGCATCGCCACCGGCCTGCTGGTGCTGAAGGATCTGGGC 1517) ATCCAGGTGGACCGGTACATCGCCTCCGAGGTGTGCGAGGATTCTATCACCGTGGGCATGGT GCGCCACCAGGGCAAGATCATGTATGTGGGCGACGTGCGGTCCGTGACACAGAAGCACATCC AGGAGTGGGGCCCATTCGATCTGGTGATCGGCGGCAGCCCCTGTAATGACCTGTCCATCGTG AACCCTGCAAGGAAGGGACTGTACGAGGGAACCGGCCGGCTGTTCTTTGAGTTTTATAGACT GCTGCACGACGCCAGGCCTAAGGAGGGCGACGATAGACCATTCTTTTGGCTGTTCGAGAATG TGGTGGCTATGGGCGTGAGCGATAAGAGGGACATCTCCAGGTTTCTGGAGTCTAACCCCGTG ATGATCGATGCAAAGGAGGTGTCCGCCGCACACAGAGCCAGGTATTTCTGGGGCAATCTGCC AGGAATGAACAGGCCACTGGCAAGCACCGTGAATGACAAGCTGGAGCTGCAGGAGTGCCTGG AGCACGGAAGGATCGCCAAGTTTTCCAAGGTGCGCACAATCACCACACGGAGCAATTCCATC AAGCAGGGCAAGGATCAGCACTTCCCCGTGTTCATGAACGAGAAGGAGGACATCCTGTGGTG TACCGAGATGGAGAGAGTGTTCGGCTTTCCAGTGCACTACACAGACGTGTCTAACATGAGCA GGCTGGCAAGGCAGCGGCTGCTGGGCAGATCTTGGAGCGTGCCCGTGATCAGGCACCTGTTC GCCCCTCTGAAGGAGTATTTTGCCTGCGTGAGCAGCGGCAACTCCAATGCCAACAGCCGGGG CCCCTCTTTCAGCTCCGGATTGGTGCCTCTGAGCCTGAGGGGCTCCCACATGGCAGCAATCC CCGCCCTGGACCCCGAGGCCGAGCCTAGCATGGACGTGATCCTGGTGGGCTCTAGCGAGCTG TCCTCTAGCGTGTCTCCAGGAACCGGAAGGGATCTGATCGCATACGAGGTGAAGGCCAATCA GCGGAACATCGAGGACATCTGTATCTGCTGTGGCAGCCTGCAGGTGCACACACAGCACCCAC TGTTCGAGGGAGGAATCTGCGCACCCTGTAAGGATAAGTTCCTGGACGCCCTGTTTCTGTAC GACGATGACGGCTACCAGTCCTATTGCTCTATCTGCTGTTCCGGCGAGACCCTGCTGATCTG CGGCAATCCAGATTGTACAAGGTGCTATTGTTTTGAGTGCGTGGACTCTCTGGTGGGACCAG GCACCAGCGGAAAGGTGCACGCCATGTCCAACTGGGTGTGCTACCTGTGCCTGCCATCCTCT CGCAGCGGACTGCTGCAGCGGAGAAGGAAGTGGAGATCCCAGCTGAAGGCCTTCTATGATAG GGAGTCTGAGAACCCCCTGGAGATGTTTGAGACCGTGCCAGTGTGGCGCCGGCAGCCCGTGA GGGTGCTGAGCCTGTTCGAGGATATCAAGAAGGAGCTGACATCCCTGGGCTTTCTGGAGTCC GGCTCTGACCCCGGACAGCTGAAGCACGTGGTGGATGTGACCGACACAGTGCGGAAGGATGT GGAGGAGTGGGGCCCTTTCGACCTGGTGTACGGAGCAACCCCTCCACTGGGACACACATGCG ACAGACCCCCTTCTTGGTACCTGTTCCAGTTTCACCGCCTGCTGCAGTATGCAAGGCCAAAG CCAGGCAGCCCTAGACCATTCTTTTGGATGTTCGTGGATAATCTGGTGCTGAACAAGGAGGA TCTGGACGTGGCCAGCAGGTTTCTGGAGATGGAGCCAGTGACCATCCCAGACGTGCACGGCG GCTCCCTGCAGAATGCCGTGCGCGTGTGGTCTAACATCCCTGCCATCAGAAGCAGGCACTGG GCACTGGTGAGCGAGGAGGAGCTGTCCCTGCTGGCCCAGAATAAGCAGAGCAGCAAGCTGGC CGCCAAGTGGCCTACAAAGCTGGTGAAGAACTGCTTCCTGCCACTGCGGGAGTACTTCAAGT ATTTTTCCACCGAGCTGACATCTAGCCTGGGAGGACCCTCCTCTGGCGCCCCACCACCTAGC GGCGGCTCCCCTGCCGGCTCTCCAACCAGCACAGAGGAGGGCACCAGCGAGTCCGCCACACC AGAGTCTGGACCTGGCACCAGCACAGAGCCATCCGAGGGCTCTGCCCCGGGCTCTCCTGCAG GCAGCCCTACCTCCACCGAAGAGGGCACCAGCACAGAGCCTTCTGAGGGCAGCGCCCCAGGC ACCTCTACAGAGCCAAGCGAGCTCGAGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCAC CAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACG GAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACA GCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCA CCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCC TGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAA CCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCA GACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAAC CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGA TGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA TCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTG AGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGAC GGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGG CACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCG CATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCC CAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCC CAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACG TGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAA AATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGG GCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAAC GAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGA GGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGC GGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAG TCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCA GCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCC AGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGC ATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCC GCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAA CACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGG GCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACG CCATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGC GACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCA AGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTG GAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTA CGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCC CACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGA AAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTC AAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGG CGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGA GCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAG TCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAA GAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGG AAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAA AAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGA GAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATAT GTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA GCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTAC AACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCT GACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGT ACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC GAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGG AGTCGACGGATCCAGCGGCTCCGAGACCCCAGGCACATCTGAGAGCGCCACCCCTGAGTCCA CCGGTAACAAAAAGCTTGAGGCCGTCGGAACCGGAATCGAACCAAAAGCAATGTCCCAGGGT TTGGTGACATTTGGCGACGTGGCTGTCGATTTTTCCCAGGAAGAGTGGGAGTGGCTCAATCC TATCCAGAGGAACTTGTACCGGAAGGTGATGCTGGAGAATTATAGAAATTTGGCATCACTGG GGTTGTGCGTTAGCAAACCAGATGTTATATCTTCCCTGGAACAGGGAAAGGAGCCCTGGAGC GCTGATTACAAAGATGATGACGATAAAGCCCCCAAGAAGAAAAGGAAGGTCCCAAAGAAAAA AAGAAAGGTGTGA Fusion Protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLG 11 Amino Acid IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV Sequence NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV NLS-NLS-3A-h3L- MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI dCas9-ZIM3-NLS- KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF NLS APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEPSMDVILVGSSEL (SEQ ID NO: SSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKFLDALFLY 1518) DDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSS RSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLES GSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPK PGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHW ALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGGPSSGAPPPS GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLED SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYID GGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTEDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSESATPESTGMNNSQGRVTFEDVTVNFTQ GEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAE KNGDIGGQIWKPKDVKESLSADYKDDDDKAPKKKRKVPKKKRKV Fusion Protein ATGGGTACCATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAATCA 11 DNA Sequence CGATCAGGAGTTCGACCCCCCTAAGGTGTACCCACCAGTGCCTGCAGAGAAGAGGAAGCCAA (SEQ ID NO: TCCGGGTGCTGAGCCTGTTTGATGGCATCGCCACCGGCCTGCTGGTGCTGAAGGATCTGGGC 1519) ATCCAGGTGGACCGGTACATCGCCTCCGAGGTGTGCGAGGATTCTATCACCGTGGGCATGGT GCGCCACCAGGGCAAGATCATGTATGTGGGCGACGTGCGGTCCGTGACACAGAAGCACATCC AGGAGTGGGGCCCATTCGATCTGGTGATCGGCGGCAGCCCCTGTAATGACCTGTCCATCGTG AACCCTGCAAGGAAGGGACTGTACGAGGGAACCGGCCGGCTGTTCTTTGAGTTTTATAGACT GCTGCACGACGCCAGGCCTAAGGAGGGCGACGATAGACCATTCTTTTGGCTGTTCGAGAATG TGGTGGCTATGGGCGTGAGCGATAAGAGGGACATCTCCAGGTTTCTGGAGTCTAACCCCGTG ATGATCGATGCAAAGGAGGTGTCCGCCGCACACAGAGCCAGGTATTTCTGGGGCAATCTGCC AGGAATGAACAGGCCACTGGCAAGCACCGTGAATGACAAGCTGGAGCTGCAGGAGTGCCTGG AGCACGGAAGGATCGCCAAGTTTTCCAAGGTGCGCACAATCACCACACGGAGCAATTCCATC AAGCAGGGCAAGGATCAGCACTTCCCCGTGTTCATGAACGAGAAGGAGGACATCCTGTGGTG TACCGAGATGGAGAGAGTGTTCGGCTTTCCAGTGCACTACACAGACGTGTCTAACATGAGCA GGCTGGCAAGGCAGCGGCTGCTGGGCAGATCTTGGAGCGTGCCCGTGATCAGGCACCTGTTC GCCCCTCTGAAGGAGTATTTTGCCTGCGTGAGCAGCGGCAACTCCAATGCCAACAGCCGGGG CCCCTCTTTCAGCTCCGGATTGGTGCCTCTGAGCCTGAGGGGCTCCCACATGGCAGCAATCC CCGCCCTGGACCCCGAGGCCGAGCCTAGCATGGACGTGATCCTGGTGGGCTCTAGCGAGCTG TCCTCTAGCGTGTCTCCAGGAACCGGAAGGGATCTGATCGCATACGAGGTGAAGGCCAATCA GCGGAACATCGAGGACATCTGTATCTGCTGTGGCAGCCTGCAGGTGCACACACAGCACCCAC TGTTCGAGGGAGGAATCTGCGCACCCTGTAAGGATAAGTTCCTGGACGCCCTGTTTCTGTAC GACGATGACGGCTACCAGTCCTATTGCTCTATCTGCTGTTCCGGCGAGACCCTGCTGATCTG CGGCAATCCAGATTGTACAAGGTGCTATTGTTTTGAGTGCGTGGACTCTCTGGTGGGACCAG GCACCAGCGGAAAGGTGCACGCCATGTCCAACTGGGTGTGCTACCTGTGCCTGCCATCCTCT CGCAGCGGACTGCTGCAGCGGAGAAGGAAGTGGAGATCCCAGCTGAAGGCCTTCTATGATAG GGAGTCTGAGAACCCCCTGGAGATGTTTGAGACCGTGCCAGTGTGGCGCCGGCAGCCCGTGA GGGTGCTGAGCCTGTTCGAGGATATCAAGAAGGAGCTGACATCCCTGGGCTTTCTGGAGTCC GGCTCTGACCCCGGACAGCTGAAGCACGTGGTGGATGTGACCGACACAGTGCGGAAGGATGT GGAGGAGTGGGGCCCTTTCGACCTGGTGTACGGAGCAACCCCTCCACTGGGACACACATGCG ACAGACCCCCTTCTTGGTACCTGTTCCAGTTTCACCGCCTGCTGCAGTATGCAAGGCCAAAG CCAGGCAGCCCTAGACCATTCTTTTGGATGTTCGTGGATAATCTGGTGCTGAACAAGGAGGA TCTGGACGTGGCCAGCAGGTTTCTGGAGATGGAGCCAGTGACCATCCCAGACGTGCACGGCG GCTCCCTGCAGAATGCCGTGCGCGTGTGGTCTAACATCCCTGCCATCAGAAGCAGGCACTGG GCACTGGTGAGCGAGGAGGAGCTGTCCCTGCTGGCCCAGAATAAGCAGAGCAGCAAGCTGGC CGCCAAGTGGCCTACAAAGCTGGTGAAGAACTGCTTCCTGCCACTGCGGGAGTACTTCAAGT ATTTTTCCACCGAGCTGACATCTAGCCTGGGAGGACCCTCCTCTGGCGCCCCACCACCTAGC GGCGGCTCCCCTGCCGGCTCTCCAACCAGCACAGAGGAGGGCACCAGCGAGTCCGCCACACC AGAGTCTGGACCTGGCACCAGCACAGAGCCATCCGAGGGCTCTGCCCCAGGCTCTCCTGCAG GCAGCCCTACCTCCACCGAAGAGGGCACCAGCACAGAGCCTTCTGAGGGCAGCGCCCCAGGC ACCTCTACAGAGCCAAGCGAGCTCGAGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCAC CAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACG GAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACA GCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCA CCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCC TGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAA CCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCA GACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAAC CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGA TGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA TCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTG AGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGAC GGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGG CACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCG CATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCC CAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCC CAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACG TGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAA AATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGG GCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAAC GAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGA GGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGC GGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAG TCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCA GCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCC AGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGC ATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCC GCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAA CACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGG GCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACG CCATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGC GACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCA AGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTG GAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTA CGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCC CACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGA AAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTC AAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGG CGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGA GCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAG TCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAA GAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGG AAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAA AAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGA GAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATAT GTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA GCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTAC AACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCT GACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGT ACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC GAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGG AGTCGACGGATCCAGCGGCTCCGAGACCCCAGGCACATCTGAGAGCGCCACCCCTGAGTCCA CCGGTATGAACAATTCACAGGGGAGAGTGACATTCGAAGACGTGACCGTGAACTTCACCCAG GGAGAATGGCAGCGCTTGAACCCAGAACAAAGGAACCTCTATCGGGACGTGATGCTGGAAAA CTACTCAAATTTGGTGAGCGTTGGGCAGGGTGAGACCACTAAGCCTGACGTGATCCTGAGAT TGGAACAGGGCAAGGAGCCTTGGCTCGAGGAAGAGGAAGTCCTGGGCTCAGGGAGGGCCGAG AAAAACGGTGATATAGGAGGCCAGATATGGAAGCCTAAGGACGTCAAGGAGAGCCTGAGCGC TGATTACAAAGATGATGACGATAAAGCCCCCAAGAAGAAAAGGAAGGTCCCAAAGAAAAAAA GAAAGGTGTGA Fusion Protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLG 12 Amino Acid IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV Sequence NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV NLS-NLS-3A-h3L- MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI dCas9-ZN627-NLS- KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF NLS APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEPSMDVILVGSSEL (SEQ ID NO: SSSVSPGTGRDLIAYEVKANQRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKELDALFLY 1520) DDDGYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPSS RSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLES GSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPK PGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHW ALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSLGGPSSGAPPPS GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLED SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYID GGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEEN EDILEDIVLTLTLFEDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQ SGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELDKAGFIKRQLV ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFF KTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKY VNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY ETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSESATPESTGDSVAFEDVAVNFTLEEWAL LDPSQKNLYRDVMRETFRNLASVGKQWEDQNIEDPFKIPRRNISHIPERLCESKEGGQGEES ADYKDDDDKAPKKKRKVPKKKRKV Fusion Protein ATGGGTACCATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAATCA 12 DNA Sequence CGATCAGGAGTTCGACCCCCCTAAGGTGTACCCACCAGTGCCTGCAGAGAAGAGGAAGCCAA (SEQ ID NO: TCCGGGTGCTGAGCCTGTTTGATGGCATCGCCACCGGCCTGCTGGTGCTGAAGGATCTGGGC 1521) ATCCAGGTGGACCGGTACATCGCCTCCGAGGTGTGCGAGGATTCTATCACCGTGGGCATGGT GCGCCACCAGGGCAAGATCATGTATGTGGGCGACGTGCGGTCCGTGACACAGAAGCACATCC AGGAGTGGGGCCCATTCGATCTGGTGATCGGCGGCAGCCCCTGTAATGACCTGTCCATCGTG AACCCTGCAAGGAAGGGACTGTACGAGGGAACCGGCCGGCTGTTCTTTGAGTTTTATAGACT GCTGCACGACGCCAGGCCTAAGGAGGGCGACGATAGACCATTCTTTTGGCTGTTCGAGAATG TGGTGGCTATGGGCGTGAGCGATAAGAGGGACATCTCCAGGTTTCTGGAGTCTAACCCCGTG ATGATCGATGCAAAGGAGGTGTCCGCCGCACACAGAGCCAGGTATTTCTGGGGCAATCTGCC AGGAATGAACAGGCCACTGGCAAGCACCGTGAATGACAAGCTGGAGCTGCAGGAGTGCCTGG AGCACGGAAGGATCGCCAAGTTTTCCAAGGTGCGCACAATCACCACACGGAGCAATTCCATC AAGCAGGGCAAGGATCAGCACTTCCCCGTGTTCATGAACGAGAAGGAGGACATCCTGTGGTG TACCGAGATGGAGAGAGTGTTCGGCTTTCCAGTGCACTACACAGACGTGTCTAACATGAGCA GGCTGGCAAGGCAGCGGCTGCTGGGCAGATCTTGGAGCGTGCCCGTGATCAGGCACCTGTTC GCCCCTCTGAAGGAGTATTTTGCCTGCGTGAGCAGCGGCAACTCCAATGCCAACAGCCGGGG CCCCTCTTTCAGCTCCGGATTGGTGCCTCTGAGCCTGAGGGGCTCCCACATGGCAGCAATCC CCGCCCTGGACCCCGAGGCCGAGCCTAGCATGGACGTGATCCTGGTGGGCTCTAGCGAGCTG TCCTCTAGCGTGTCTCCAGGAACCGGAAGGGATCTGATCGCATACGAGGTGAAGGCCAATCA GCGGAACATCGAGGACATCTGTATCTGCTGTGGCAGCCTGCAGGTGCACACACAGCACCCAC TGTTCGAGGGAGGAATCTGCGCACCCTGTAAGGATAAGTTCCTGGACGCCCTGTTTCTGTAC GACGATGACGGCTACCAGTCCTATTGCTCTATCTGCTGTTCCGGCGAGACCCTGCTGATCTG CGGCAATCCAGATTGTACAAGGTGCTATTGTTTTGAGTGCGTGGACTCTCTGGTGGGACCAG GCACCAGCGGAAAGGTGCACGCCATGTCCAACTGGGTGTGCTACCTGTGCCTGCCATCCTCT CGCAGCGGACTGCTGCAGCGGAGAAGGAAGTGGAGATCCCAGCTGAAGGCCTTCTATGATAG GGAGTCTGAGAACCCCCTGGAGATGTTTGAGACCGTGCCAGTGTGGCGCCGGCAGCCCGTGA GGGTGCTGAGCCTGTTCGAGGATATCAAGAAGGAGCTGACATCCCTGGGCTTTCTGGAGTCC GGCTCTGACCCCGGACAGCTGAAGCACGTGGTGGATGTGACCGACACAGTGCGGAAGGATGT GGAGGAGTGGGGCCCTTTCGACCTGGTGTACGGAGCAACCCCTCCACTGGGACACACATGCG ACAGACCCCCTTCTTGGTACCTGTTCCAGTTTCACCGCCTGCTGCAGTATGCAAGGCCAAAG CCAGGCAGCCCTAGACCATTCTTTTGGATGTTCGTGGATAATCTGGTGCTGAACAAGGAGGA TCTGGACGTGGCCAGCAGGTTTCTGGAGATGGAGCCAGTGACCATCCCAGACGTGCACGGCG GCTCCCTGCAGAATGCCGTGCGCGTGTGGTCTAACATCCCTGCCATCAGAAGCAGGCACTGG GCACTGGTGAGCGAGGAGGAGCTGTCCCTGCTGGCCCAGAATAAGCAGAGCAGCAAGCTGGC CGCCAAGTGGCCTACAAAGCTGGTGAAGAACTGCTTCCTGCCACTGCGGGAGTACTTCAAGT ATTTTTCCACCGAGCTGACATCTAGCCTGGGAGGACCCTCCTCTGGCGCCCCACCACCTAGC GGCGGCTCCCCTGCCGGCTCTCCAACCAGCACAGAGGAGGGCACCAGCGAGTCCGCCACACC AGAGTCTGGACCTGGCACCAGCACAGAGCCATCCGAGGGCTCTGCCCCAGGCTCTCCTGCAG GCAGCCCTACCTCCACCGAAGAGGGCACCAGCACAGAGCCTTCTGAGGGCAGCGCCCCAGGC ACCTCTACAGAGCCAAGCGAGCTCGAGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCAC CAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGG TGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACG GAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACA GCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCA CCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCC TGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAA CCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCA GACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAAC CTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGA TGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGA TCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTG AGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGC CTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGAC GGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGG CACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAG GAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCG CATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAA AGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCC CAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCC CAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACG TGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAA AATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGG GCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAAC GAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGA GGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGC GGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAG TCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCA GCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCC AGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGC ATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCC GCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAA CACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGG GCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACG CCATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGC GACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCA AGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTG GAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTA CGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCC CACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGA AAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTC AAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGG CGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGA GCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAG TCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAA GAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGG AAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAA AAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGA GAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATAT GTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGA GCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA GCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTAC AACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCT GACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGT ACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC GAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGG AGTCGACGGATCCAGCGGCTCCGAGACCCCAGGCACATCTGAGAGCGCCACCCCTGAGTCCA CCGGTGACTCCGTTGCTTTCGAGGACGTGGCCGTGAACTTCACACTTGAGGAATGGGCCTTG CTCGACCCAAGTCAGAAGAATCTGTACAGAGACGTGATGCGGGAGACATTCAGGAATCTCGC CAGTGTCGGAAAGCAGTGGGAAGACCAGAACATCGAAGATCCTTTCAAGATACCACGGCGCA ATATCTCCCACATTCCTGAGAGGCTGTGTGAATCTAAGGAAGGCGGACAAGGTGAGGAAAGC GCTGATTACAAAGATGATGACGATAAAGCCCCCAAGAAGAAAAGGAAGGTCCCAAAGAAAAA AAGAAAGGTGTGA Fusion Protein MGTMNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDS 13 Amino Acid ITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLF Sequence FEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARY 3A-3L-NLS-dCas9- FWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEK NLS-KOX1KRAB EDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGNS (SEQ ID NO: NANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLERNIDKVLKSLGFLESG 1525) SGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALP RQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKH APLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPPS GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEPKKKRKVYMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNL IGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEE DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIE GDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEK KNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEV VDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKD FLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLI NGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSID NKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAG FIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANL DKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH QSITGLYETRIDLSQLGGDPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEW KLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEP Fusion Protein ATGGGTACCATGAACCATGACCAGGAATTTGACCCCCCAAAGGTTTACCCACCTGTGCCAGC 13 DNA Sequence TGAGAAGAGGAAGCCCATCCGCGTGCTGTCTCTCTTTGATGGGATTGCTACAGGGCTCCTGG (SEQ ID NO: TGCTGAAGGACCTGGGCATCCAAGTGGACCGCTACATTGCCTCCGAGGTGTGTGAGGACTCC 1526) ATCACGGTGGGCATGGTGCGGCACCAGGGAAAGATCATGTACGTCGGGGACGTCCGCAGCGT CACACAGAAGCATATCCAGGAGTGGGGCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCA ATGACCTCTCCATTGTCAACCCTGCCCGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTC TTTGAGTTCTACCGCCTCCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTT CTGGCTCTTTGAGAATGTGGTGGCCATGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTC TTGAGTCTAACCCCGTGATGATTGACGCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTAC TTCTGGGGTAACCTTCCTGGCATGAACAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGA GCTGCAAGAGTGTCTGGAGCACGGCAGAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCA CCAGGTCAAACTCTATAAAGCAGGGCAAAGACCAGCATTTCCCCGTCTTCATGAACGAGAAG GAGGACATCCTGTGGTGCACTGAAATGGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGA CGTCTCCAACATGAGCCGCTTGGCGAGGCAGAGACTGCTGGGCCGATCGTGGAGCGTGCCGG TCATCCGCCACCTCTTCGCTCCGCTGAAGGAATATTTTGCTTGTGTGTCTAGCGGCAATAGT AACGCTAACAGCCGCGGGCCGAGCTTCAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAG CCATATGGGCCCTATGGAGATATACAAGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGG TACTGAGCCTCTTCAGAAACATCGACAAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGT TCTGGTTCTGGGGGAGGAACGCTGAAGTACGTGGAAGATGTCACAAATGTCGTGAGGAGAGA CGTGGAGAAATGGGGCCCCTTTGACCTGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTT GTGATCGCTGTCCCGGCTGGTACATGTTCCAGTTCCACCGGATCCTGCAGTATGCGCTGCCT CGCCAGGAGAGTCAGCGGCCCTTCTTCTGGATATTCATGGACAATCTGCTGCTGACTGAGGA TGACCAAGAGACAACTACCCGCTTCCTTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTG GCAGAGACTACCAGAATGCTATGCGGGTGTGGAGCAACATTCCAGGGCTGAAGAGCAAGCAT GCGCCCCTGACCCCAAAGGAAGAAGAGTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCT GGACGCCCCGAAAGTTGACCTCCTGGTGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCA AGTATTTTTCTCAAAACTCACTTCCTCTTGGAGGGCCGAGCTCTGGCGCACCCCCACCAAGT GGAGGGTCTCCTGCCGGGTCCCCAACATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCC CGAGTCAGGCCCTGGTACCTCCACAGAACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTG GAAGCCCTACTTCCACCGAAGAAGGCACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGG ACCAGCACTGAACCATCTGAGCCAAAAAAGAAGAGAAAGGTATACATGGACAAGAAGTACAG CATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGG TGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTG ATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGC CAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACG AGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAG GATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGA GAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACC TGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAG GGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTA CAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGT CTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAG AAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAG CAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACC TGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAAC CTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCC CCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAG CTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAAC GGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCC CATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGC TGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTG CACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGAT CGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCA GATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTG GTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCT GCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACG AGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAG CAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCT GAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAG ATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGAC TTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTT TGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAG TGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATC AACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTT CGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCC AGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGC AGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGT GATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCC AGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTG GGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTA CCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGC TGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGAT AACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGA GGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGAATGCCAAGCTGATTACCCAGA GGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGC TTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGA CTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGATCCGGGAAGTGAAAGTGATCA CCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAG ATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGAT CAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGC GGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTAC AGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCG GCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGTGGGATAAGGGCCGGGACTTTG CCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAG ACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGACAAGCTGATCGCCAG AAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTG TGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTG CTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGC CAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCG AGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAA CTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAA GGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAACACTACCTGG ACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTG GACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCCTATCAGAGAGCAGGCCGAGAA TATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACA CCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCAC CAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACCCAAA AAAGAAGAGAAAGGTAAGCGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAA GCACCGGTCGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGG AAGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAA CCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAG AAGAGCCCTGA Fusion Protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLG 14 Amino Acid IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV Sequence NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV NLS-NLS-3A-3L- MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI dCas9-ZIM3-NLS- KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF NLS APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLER (SEQ ID NO: NIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPG 1527) WYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQN AMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLI AQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYAD LFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLL KIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSF LKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGL SELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNI VKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAG ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYEDTTIDRKRYTSTKEV LDATLIHQSITGLYETRIDLSQLGGDSGSETPGTSESATPESTGMNNSQGRVTFEDVTVNFT QGEWQRLNPEQRNLYRDVMLENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRA EKNGDIGGQIWKPKDVKESLSADYKDDDDKAPKKKRKVPKKKRKV Fusion Protein ATGGGTACCATGCCAAAAAAGAAGAGAAAGGTACCGAAGAAAAAAAGAAAGGTATACAACCA 14 DNA Sequence TGACCAGGAATTCGACCCCCCAAAGGTTTACCCACCTGTGCCAGCTGAGAAGAGGAAGCCCA (SEQ ID NO: TCCGCGTGCTGTCTCTCTTTGATGGGATTGCTACAGGGCTCCTGGTGCTGAAGGACCTGGGC 1528) ATCCAAGTGGACCGCTACATTGCCTCCGAGGTGTGTGAGGACTCCATCACGGTGGGCATGGT GCGGCACCAGGGAAAGATCATGTACGTCGGGGACGTCCGCAGCGTCACACAGAAGCATATCC AGGAGTGGGGCCCATTCGACCTGGTGATTGGAGGCAGTCCCTGCAATGACCTCTCCATTGTC AACCCTGCCCGCAAGGGACTTTATGAGGGTACTGGCCGCCTCTTCTTTGAGTTCTACCGCCT CCTGCATGATGCGCGGCCCAAGGAGGGAGATGATCGCCCCTTCTTCTGGCTCTTTGAGAATG TGGTGGCCATGGGCGTTAGTGACAAGAGGGACATCTCGCGATTTCTTGAGTCTAACCCCGTG ATGATTGACGCCAAAGAAGTGTCTGCTGCACACAGGGCCCGTTACTTCTGGGGTAACCTTCC TGGCATGAACAGGCCTTTGGCATCCACTGTGAATGATAAGCTGGAGCTGCAAGAGTGTCTGG AGCACGGCAGAATAGCCAAGTTCAGCAAAGTGAGGACCATTACCACCAGGTCAAACTCTATA AAGCAGGGCAAAGACCAGCATTTCCCCGTCTTCATGAACGAGAAGGAGGACATCCTGTGGTG CACTGAAATGGAAAGGGTGTTTGGCTTCCCCGTCCACTACACAGACGTCTCCAACATGAGCC GCTTGGCGAGGCAGAGACTGCTGGGCCGATCGTGGAGCGTGCCGGTCATCCGCCACCTCTTC GCTCCGCTGAAGGAATATTTTGCTTGTGTGTCTAGCGGCAATAGTAACGCTAACAGCCGCGG GCCGAGCTTCAGCAGCGGCCTGGTGCCGTTAAGCTTGCGCGGCAGCCATATGGGCCCTATGG AGATATACAAGACAGTGTCTGCATGGAAGAGACAGCCAGTGCGGGTACTGAGCCTCTTCAGA AACATCGACAAGGTACTAAAGAGTTTGGGCTTCTTGGAAAGCGGTTCTGGTTCTGGGGGAGG AACGCTGAAGTACGTGGAAGATGTCACAAATGTCGTGAGGAGAGACGTGGAGAAATGGGGCC CCTTTGACCTGGTGTACGGCTCGACGCAGCCCCTAGGCAGCTCTTGTGATCGCTGTCCCGGC TGGTACATGTTCCAGTTCCACCGGATCCTGCAGTATGCGCTGCCTCGCCAGGAGAGTCAGCG GCCCTTCTTCTGGATATTCATGGACAATCTGCTGCTGACTGAGGATGACCAAGAGACAACTA CCCGCTTCCTTCAGACAGAGGCTGTGACCCTCCAGGATGTCCGTGGCAGAGACTACCAGAAT GCTATGCGGGTGTGGAGCAACATTCCAGGGCTGAAGAGCAAGCATGCGCCCCTGACCCCAAA GGAAGAAGAGTATCTGCAAGCCCAAGTCAGAAGCAGGAGCAAGCTGGACGCCCCGAAAGTTG ACCTCCTGGTGAAGAACTGCCTTCTCCCGCTGAGAGAGTACTTCAAGTATTTTTCTCAAAAC TCACTTCCTCTTGGAGGGCCGAGCTCTGGCGCACCCCCACCAAGTGGAGGGTCTCCTGCCGG GTCCCCAACATCTACTGAAGAAGGCACCAGCGAATCCGCAACGCCCGAGTCAGGCCCTGGTA CCTCCACAGAACCATCTGAAGGTAGTGCGCCTGGTTCCCCAGCTGGAAGCCCTACTTCCACC GAAGAAGGCACGTCAACCGAACCAAGTGAAGGATCTGCCCCTGGGACCAGCACTGAACCATC TGAGATGGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCG TGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGG CACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGAGAAACAGCCGAGGC CACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATC TGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAA GAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGT GGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGG ACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTC CGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTT CATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCG TGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATC GCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGG CCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCA AGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGAC CTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAA CACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACC AGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATT TTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGATGGCGGAGCCAGCCAGGAAGA GTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGA AGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCAC CAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCT GAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCC CTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACC CCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGAT GACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACG AGTACTTCACCGTGTACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAG CCCGCCTTCCTGAGCGGCGAGCAGAAAAAAGCCATCGTGGACCTGCTGTTCAAGACCAACCG GAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCG TGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTG AAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATAT CGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATG CCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGC AGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGA TTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC TGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAG CACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGT GGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGG CCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATC GAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCA GCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACC AGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACGCTATCGTGCCTCAGAGCTTT CTGAAGGACGACTCCATCGATAACAAAGTGCTGACTCGGAGCGACAAGAACCGGGGCAAGAG CGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGCCAGCTGCTGA ATGCCAAGCTGATTACCCAGAGGAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTG AGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAA GCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAACGACAAACTGA TCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTC CAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGC CGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCG ACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCT ACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGC CAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACAGGCGAGATCGTGT GGGATAAGGGCCGGGACTTTGCCACCGTGCGGAAAGTGCTGTCTATGCCCCAAGTGAATATC GTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAA CAGCGACAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACA GCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAA CTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAA TCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGC TGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGC GAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGC CAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGG AACAGCACAAACACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTG ATCCTGGCCGACGCTAATCTGGACAAGGTGCTGAGCGCCTACAACAAGCACAGAGACAAGCC TATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTG CCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTG CTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTC TCAGCTGGGAGGCGACAGCGGAAGTGAGACCCCAGGTACATCCGAATCAGCAACGCCTGAAA GCACCGGTATGAACAATTCACAGGGGAGAGTGACATTCGAAGACGTGACCGTGAACTTCACC CAGGGAGAATGGCAGCGCTTGAACCCAGAACAAAGGAACCTCTATCGGGACGTGATGCTGGA AAACTACTCAAATTTGGTGAGCGTTGGGCAGGGTGAGACCACTAAGCCTGACGTGATCCTGA GATTGGAACAGGGCAAGGAGCCTTGGCTCGAGGAAGAGGAAGTCCTGGGCTCAGGGAGGGCC GAGAAAAACGGTGATATAGGAGGCCAGATATGGAAGCCTAAGGACGTCAAGGAGAGCCTGAG CGCTGATTACAAAGATGATGACGATAAAGCCCCAAAAAAGAAGAGAAAGGTACCGAAGAAAA AAAGAAAGGTCTGA

Sequences from FIG. 14A can be found below:

Description Sequence SEQ ID NO: gRNA009 ACUGCCUGGCUCACUCCUCC 1270 gRNA003 UGCGGAAACCUUCUAGGGUG 1264 gRNA093 AACCUGAUCCUCCAGUCCGG 1354 gRNA011 AUCGUCCGAUGGGGCUCUGG 1272 gRNA007 GGUGUGGGUGCUUGACGCCU 1268 gRNA077 CAAAUCCUAACUGGGCUGGA 1338 gRNA113 GGGCGCCGCCGUUCAGUUCA 1374 gRNA004 GCGGAAACCUUCUAGGGUGU 1265 gRNA008 GUGUGGGUGCUUGACGCCUG 1269 gRNA012 AGGAUCGUCCGAUGGGGCUC 1273 gRNA111 AGUUCAGGGUCUGAGCCUGG 1372 gRNA005 UCAAGCACCCACACCCUAGA 1266 gRNA013 UCAGAUAGGAUCGUCCGAUG 1274

Sequences from FIG. 15 can be found below:

ZFoff-15 Target GATGGGGCTCTGGTGGCG (SEQ ID NO: 676) Sequence ZFoff-15 ZFP SRPGERPFQCRICMRNFSRTDTLARHLRTHTGEKPFQCRICMRNFSRKTALNRHLKTHTGSQ Amino Acid KPFQCRICMRNFSRNESLKVHLRTHTGEKPFQCRICMRNFSVKNTLTRHLKTHTGSQKPFQC Sequence RICMRNFSRREHLVRHLRTHTGEKPFQCRICMRNFSLTHNLRRHLKTHLRGS (SEQ ID NO: 677) ZFoff-15 Full MGTMYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCED Amino Acid SITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRL Sequence FFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRAR YFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNE KEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGN SNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLES GSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYAL PRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSK HAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPP SGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEPKKKRKVSRPGERPFQCRICMRNFSRTDTLARHLRTHTGEKPFQCRICMRNFSR KTALNRHLKTHTGSQKPFQCRICMRNFSRNESLKVHLRTHTGEKPFQCRICMRNFSVKNTLT RHLKTHTGSQKPFQCRICMRNFSRREHLVRHLRTHTGEKPFQCRICMRNESLTHNLRRHLKT HLRGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNV MLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKA (SEQ ID NO: 678) ZFoff-15 with MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLG 2xNLS IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLER NIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPG WYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQN AMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSESRPGERPFQCRICMRNFSRTDTLARHLRTHTGEKPFQCRI CMRNFSRKTALNRHLKTHTGSQKPFQCRICMRNFSRNESLKVHLRTHTGEKPFQCRICMRNF SVKNTLTRHLKTHTGSQKPFQCRICMRNFSRREHLVRHLRTHTGEKPFQCRICMRNFSLTHN LRRHLKTHLRGSSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNV MLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKKRKV (SEQ ID NO: 679) ZFoff-60 Target GCAGAGGCCGGAGGGGGTC (SEQ ID NO: 740) Sequence ZFoff-60 ZFP SRPGERPFQCRICMRNFSDPSVLKRHLRTHTGEKPFQCRICMRNFSRTEHLARHLKTHTGGG Amino Acid GSQKPFQCRICMRNFSQSPHLKRHLRTHTGEKPFQCRICMRNFSDQTTLRRHLKTHTGSQKP Sequence FQCRICMRNFSKHSNLTRHTRTHTGEKPFQCRICMRNFSQMETLKRHLRTHLRGS (SEQ ID NO: 680) ZFoff-60 Full MGTMYNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGIQVDRYIASEVCED Amino Acid SITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRL Sequence FFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRAR YFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNE KEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGN SNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLES GSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYAL PRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSK HAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPP SGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEPKKKRKVSRPGERPFQCRICMRNFSDPSVLKRHLRTHTGEKPFQCRICMRNFSR TEHLARHLKTHTGGGGSQKPFQCRICMRNFSQSPHLKRHLRTHTGEKPFQCRICMRNFSDQT TLRRHLKTHTGSQKPFQCRICMRNFSKHSNLTRHTRTHTGEKPFQCRICMRNFSQMETLKRH LRTHLRGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVY RNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKA (SEQ ID NO: 681) ZFoff-60 with MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLG 2xNLS IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLER NIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPG WYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQN AMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSESRPGERPFQCRICMRNFSDPSVLKRHLRTHTGEKPFQCRI CMRNFSRTEHLARHLKTHTGGGGSQKPFQCRICMRNFSQSPHLKRHLRTHTGEKPFQCRICM RNFSDQTTLRRHLKTHTGSQKPFQCRICMRNFSKHSNLTRHTRTHTGEKPFQCRICMRNFSQ METLKRHLRTHLRGSSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVY RNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKKRKV (SEQ ID NO: 682) ZFoff-137 Target GGTGGGGAGGACTGTGCA (SEQ ID NO: 683) Sequence ZFoff-137 ZFP SRPGERPFQCRICMRNFSQSTTLKRHLRTHTGEKPFQCRICMRNFSGAHGLAGHLKTHTGSQ Amino Acid KPFQCRICMRNFSDQTNLRRHLRTHTGEKPFQCRICMRNFSRQDNLQRHLKTHTGSQKPFQC Sequence RICMRNFSKGDHLRRHTRTHTGEKPFQCRICMRNFSEAHHLSRHLRTHLRGS (SEQ ID NO: 684) ZFoff-137 Full MGTMYNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGIQVDRYIASEVCED Amino Acid SITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRL Sequence FFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRAR YFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNE KEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGN SNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLES GSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYAL PRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSK HAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPP SGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEPKKKRKVSRPGERPFQCRICMRNFSQSTTLKRHLRTHTGEKPFQCRICMRNESG AHGLAGHLKTHTGSQKPFQCRICMRNFSDQTNLRRHLRTHTGEKPFQCRICMRNFSRQDNLQ RHLKTHTGSQKPFQCRICMRNFSKGDHLRRHTRTHTGEKPFQCRICMRNFSEAHHLSRHLRT HLRGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNV MLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKA (SEQ ID NO: 685) ZFoff-137 with MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLG 2xNLS IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLER NIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPG WYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQN AMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSESRPGERPFQCRICMRNFSQSTTLKRHLRTHTGEKPFQCRI CMRNFSGAHGLAGHLKTHTGSQKPFQCRICMRNFSDQTNLRRHLRTHTGEKPFQCRICMRNF SRQDNLQRHLKTHTGSQKPFQCRICMRNFSKGDHLRRHTRTHTGEKPFQCRICMRNESEAHH LSRHLRTHLRGSSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNV MLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKKRKV (SEQ ID NO: 686) ZFoff-152 Target GCAGGAGGACGAGGACGGC (SEQ ID NO: 687) Sequence ZFoff-152 ZFP SRPGERPFQCRICMRNFSTPSKLDRHTRTHTGEKPFQCRICMRNFSLAENLRRHLRTHTGSQ Amino Acid KPFQCRICMRNFSRQDNLGRHLRTHTGEKPFQCRICMRNFSDGGNLGRHLKTHTGGGGSQKP Sequence FQCRICMRNFSQSPHLKRHLRTHTGEKPFQCRICMRNFSQSTSLQRHLKTHLRGS (SEQ ID NO: 688) ZFoff-152 Full MGTMYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCED Amino Acid SITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRL Sequence FFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRAR YFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNE KEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGN SNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLES GSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYAL PRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSK HAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPP SGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEPKKKRKVSRPGERPFQCRICMRNFSTPSKLDRHTRTHTGEKPFQCRICMRNFSL AENLRRHLRTHTGSQKPFQCRICMRNFSRQDNLGRHLRTHTGEKPFQCRICMRNFSDGGNLG RHLKTHTGGGGSQKPFQCRICMRNFSQSPHLKRHLRTHTGEKPFQCRICMRNFSQSTSLQRH LKTHLRGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVY RNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKA (SEQ ID NO: 689) ZFoff-152 with MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLG 2xNLS IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLER NIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPG WYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQN AMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSESRPGERPFQCRICMRNFSTPSKLDRHTRTHTGEKPFQCRI CMRNFSLAENLRRHLRTHTGSQKPFQCRICMRNFSRQDNLGRHLRTHTGEKPFQCRICMRNF SDGGNLGRHLKTHTGGGGSQKPFQCRICMRNFSQSPHLKRHLRTHTGEKPFQCRICMRNFSQ STSLQRHLKTHLRGSSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVY RNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKKRKV (SEQ ID NO: 690) ZFoff-153 Target GCAGGAGGACGAGGACGGC (SEQ ID NO: 691) Sequence ZFoff-153 ZFP SRPGERPFQCRICMRNFSERAKLIRHLRTHTGEKPFQCRICMRNFSDPSNLRRHLKTHTGSQ Amino Acid KPFQCRICMRNFSRQDNLGRHLRTHTGEKPFQCRICMRNFSDQGNLGRHLKTHTGGGGSQKP Sequence FQCRICMRNFSQSAHLKRHLRTHTGEKPFQCRICMRNFSQDVSLVRHLKTHLRGS (SEQ ID NO: 692) ZFoff-153 Full MGTMYNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCED Amino Acid SITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRL Sequence FFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRAR YFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNE KEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKEYFACVSSGN SNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLES GSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYAL PRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSK HAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPLGGPSSGAPPP SGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEPKKKRKVSRPGERPFQCRICMRNFSERAKLIRHLRTHTGEKPFQCRICMRNFSD PSNLRRHLKTHTGSQKPFQCRICMRNFSRQDNLGRHLRTHTGEKPFQCRICMRNFSDQGNLG RHLKTHTGGGGSQKPFQCRICMRNFSQSAHLKRHLRTHTGEKPFQCRICMRNFSQDVSLVRH LKTHLRGSPKKKRKVSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVY RNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKA (SEQ ID NO: 693) ZFoff-153 with MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLG 2xNLS IQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIV NPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPV MIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSI KQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLF APLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMGPMEIYKTVSAWKRQPVRVLSLER NIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPG WYMFQFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQN AMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLLPLREYFKYFSQN SLPLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSESRPGERPFQCRICMRNFSERAKLIRHLRTHTGEKPFQCRI CMRNFSDPSNLRRHLKTHTGSQKPFQCRICMRNFSRQDNLGRHLRTHTGEKPFQCRICMRNF SDQGNLGRHLKTHTGGGGSQKPFQCRICMRNFSQSAHLKRHLRTHTGEKPFQCRICMRNFSQ DVSLVRHLKTHLRGSSGSETPGTSESATPESTGRTLVTFKDVFVDFTREEWKLLDTAQQIVY RNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKKRKV (SEQ ID NO: 694)

Cell Culture and Transfection

HeLa (ATCC-CRM-CCL-2), Hepa1-6 (PCSK9-IRES-TdTomato), Huh7 (Sekisui XenoTech, LLC) and HEK293T Griptite (CLTA-GFP) cells were cultured in DMEM with 10% FBS. All experiments in HeLa and Huh7 cells were done using chemically synthesized guide RNA and in vitro transcribed effector construct. HeLa cells were reverse transfected using TransIT-X2 transfection reagent from Mirus (Cat #MIR6003). Huh7 cells were reverse transfected using MessengerMAX reagent from Invitrogen (Cat #LMRNA003). Secreted PCSK9 levels were measured at the indicated time points using LEGEND MAX™ Human PCSK9 ELISA Kit from Biolegend (Cat #443107). All ELISA data was normalized for cell numbers using CellTiter-Glo kit from Promega (Cat #G7571).

HEK293T Griptite cells with GFP knocked into the CLTA locus as an in-frame CLTA fusion were co-transfected with plasmids encoding effector construct and human CLTA guide RNA using TransIT-X2 transfection reagent from Mirus (Cat #MIR6003). GFP was measured by FACS for GFP expression as a surrogate for CLTA expression.

Hepa1-6 cells were co-transfected with plasmids encoding effector construct and mouse PCSK9 guide RNA using SF Cell Line 96-well Nucleofector Kit (Cat #V4SC-2096, program code: CM-138) in Amaxa 4D nucleofector device from Lonza. At the indicated timepoint, cells were FACS analyzed for TdTomato expression as a surrogate for PCSK9 levels.

In Vitro Transcription of Effector Constructs and Synthetic gRNA

1 μg of linearized effector template was used to set up in-vitro transcription reactions using T7 mScript™ Standard mRNA Production System from CellScript (Cat #C-MSC100625) according to manufacturer's instructions to obtain RNA that had a Cap 1 structure on the 5′ end and was 3′polyadenylated. End-modified sgRNA that had three 2′O-methyl modified nucleotides with phosphorothioate linkages on both 5′ and 3′ ends were obtained from Integrated DNA Technologies.

Methylation Profiling

Genomic DNA was extracted from each well of a 96-well culture plate using a DNAdvance DNA Extraction from Tissue Kit (Beckman Coulter). After quantification of genomic DNA via High-Sensitivity DNA 1× kit (Quant-IT), each genomic DNA sample was bisulfite converted using an EZ-96 DNA Methylation-Gold MagPrep kit (Zymo Research) according to manufacturer's instructions. For hybridization capture experiments, DNA libraries were prepared using the xGen™ Methyl-Seq DNA Library Prep Kit (IDT) and hybrid capture was conducted using the xGen™ Hybridization Capture of DNA libraries kit (IDT). For amplicon sequencing experiments, DNA libraries were prepared using the xGen™ Methyl-Seq DNA Library Prep Kit (IDT) and hybrid capture was conducted using the xGen™ Hybridization Capture of DNA libraries (IDT). Bisulfite-converted DNA from each sample was used to seed PCR corresponding to each of the two VIM amplicons using a Platinum Taq kit (Invitrogen). Pooled products were cleaned using the AMPure XP kit (Beckman Coulter) and fragment size assessment via D1000 screentape on a Tapestation 4200 (Agilent) prior to sequencing by commercial service (Azenta).

Example 13: Bacterial DNA Methyltransferases

In this experiment, a panel of bacterial proteins were screened for DNA methyltransferase activity in mammalian cells. These bacterial DNA methyltransferases (Table 12) were tested for epigenetic silencing activity by fusing them N-terminally to a dCas9 domain using the experimental procedure of Example 1. These constructs were then transfected in a reporter cell line that expresses GFP under the control of the mammalian promoter of CTLA4.

TABLE 12 Bacterial DNA methyltransferases Description Sequence M.AluI MSKANAKYSFVDLFAGIGGFHAALAATGGVCEYAVEIDREAAAVYERNWNKPALGDITDDAN methyltransferase DEGVTLRGYDGPIDVLTGGFPCQPFSKSGAQHGMAETRGTLFWNIARIIEEREPTVLILENV RNLVGPRHRHEWLTIIETLRFFGYEVSGAPAIFSPHLLPAWMGGTPQVRERVFITATLVPER MRDERSTIRRATGRPLEGFPYWADSWTDFRELSRLVVIRGFQAPEREVVGDRKRYVARTDMP EGFVPASVTRPAIDETLPAWKQSHLRRNYDFFERHFAEVVAWAYRWGVYTDLFPASRRKLEW QAQDAPRLWDTVMHFRPSGIRAKRPTYLPALVAITQTSIVGPLERRLSPRETARLQGLPEWF DFGEQRAAATYKQMGNGVNVGVVRHILREHVRRDRALLKLTPAGQRIINAVLADEPDATVGA LGAAE (SEQ ID NO: 675) M.MspI MKPEILKLIRSKLDLTQKQASEIIEVSDKTWQQWESGKTEMHPAYYSFLQEKLKDKINFEEL methyltransferase SAQKTLQKKIFDKYNQNQITKNAEELAEITHIEERKDAYSSDFKFIDLESGIGGIRQSFEVN GGKCVFSSEIDPFAKFTYYTNFGVVPFGDITKVEATTIPQHDILCAGFPCQPFSHIGKREGF EHPTQGTMFHEIVRIIETKKTPVLFLENVPGLINHDDGNTLKVIIETLEDMGYKVHHTVLDA SHFGIPQKRKRFYLVAFLNQNIHFEFPKPPMISKDIGEVLESDVTGYSISEHLQKSYLFKKD DGKPSLIDKNTTGAVKTLVSTYHKIQRLTGTFVKDGETGIRLLTTNECKAIMGFPKDFVIPV SRTQMYRQMGNSVVVPVVTKIAEQISLALKTVNQQSPQENFELELV (SEQ ID NO: 610) M.HaeIII MNLISLFSGAGGLDLGFQKAGFRIICANEYDKSIWKTYESNHSAKLIKGDISKISSDEFPKC methyltransferase DGIIGGPPCQSWSEGGSLRGIDDPRGKLFYEYIRILKQKKPIFFLAENVKGMMAQRHNKAVQ EFIQEFDNAGYDVHIILLNANDYGVAQDRKRVFYIGFRKELNINYLPPIPHLIKPTFKDVIW DLKDNPIPALDKNKTNGNKCIYPNHEYFIGSYSTIFMSRNRVRQWNEPAFTVQASGRQCQLH PQAPVMLKVSKNLNKFVEGKEHLYRRLTVRECARVQGFPDDFIFHYESLNDGYKMIGNAVPV NLAYEIAKTIKSALEICKGN (SEQ ID NO: 608) M. HhaI MIEIKDKQLTGLRFIDLFAGLGGFRLALESCGAECVYSNEWDKYAQEVYEMNFGEKPEGDIT methyltransferase QVNEKTIPDHDILCAGFPCQAFSISGKQKGFEDSRGTLFFDIARIVREKKPKVVEMENVKNF ASHDNGNTLEVVKNTMNELDYSFHAKVLNALDYGIPQKRERIYMICFRNDLNIQNFQFPKPF ELNTFVKDLLLPDSEVEHLVIDRKDLVMTNQEIEQTTPKTVRLGIVGKGGQGERIYSTRGIA ITLSAYGGGIFAKTGGYLVNGKTRKLHPRECARVMGYPDSYKVHPSTSQAYKQFGNSVVINV LQYIAYNIGSSLNFKPY (SEQ ID NO: 609) M. SssI MSKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKL methyltransferase EYKSVSREEMIDYLENKTLSWNSKNPVSNGYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDL YKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRGSGTRSGLLWEIERALDSTEKNDLPKYLLME NVGALLHKKNEEELNQWKQKLESLGYQNSIEVLNAADFGSSQARRRVFMISTLNEFVELPKG DKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKSNINKASLIGYSKENSEGYVYDPEFT GPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDSQDGKRVNEIEFLTENQKIFVCGNSI SVEVLEAIIDKIGG (SEQ ID NO: 605)

M.SssI DNA methyltransferase was able to efficiently methylate DNA in mammalian cells (FIG. 16) with stable silencing up to 30 days. Sequences from FIG. 16 can be found below:

Description Sequence SEQ ID NO: TAR087 Target DNA Sequence GGCGGAAGTGCTGCCTGATA 667 TAR089 Target DNA Sequence GCAACACCGCCTAGACCGAC 668 TAR090 Target DNA Sequence GGAGGTTCAGAAAGCCTAAG 669 TAR091 Target DNA Sequence GCAGCACTAGAGTCCCCTCA 670 TAR092 Target DNA Sequence GAACAACTCGTGACTGGGGT 671 TAR093 Target DNA Sequence GGGAGAGGAGGACAAGCGCC 672 TAR094 Target DNA Sequence GGACCCACCGACACCACGCC 673 TAR114 Target DNA Sequence CGCGGGAAAATGCAAGACGA 674

The methylation profile of these cells was also analyzed at day 29 confirming a 20% methylation of the target gene (FIGS. 17-18).

Another three orthologous DNA methyltransferases, predicted to be closely related to M. SssI, are identified and tested for epigenetic silencing activity using the experimental procedures of Example 1 (Table 13).

TABLE 13 Bacterial methyltransferases Description SEQ ID NO DNA cytosine methyltransferase 601 Mycoplasmatales bacterium DNA cytosine methyltransferase 602 Mycoplasma marinum DNA (cytosine-5-)-methyltransferase 603 Spiroplasma chinense

The DNA methyltransferases of Table 13 are predicted to have similar or improved function to M. SssI. Sequences are tested in the context of CRISPR-off, in place of murine DNMT3A/DNMT3L, and their function is compared with the function of M. SssI DNA methyltransferase in silencing the PCSK9 locus in a HeLa TdTomato system, to identify novel characteristics and improved function.

Example 14: Alternative KRAB Domains

In this example, fusion proteins were constructed with alternative KRAB domains (Table 14) and showed improved activity as compared to CRISPR-off when tested using the experimental procedures of Example 1 (FIGS. 19A-19D).

TABLE 14 Alternative KRAB Domains Description Sequence ZFP28 MKKLEAVGTGIEPKAMSQGLVTFGDVAVDFSQEEWEWLNPIQRNLYRKVMLENYRNLASLGL CVSKPDVISSLEQGKEPWTVKRKMTRAWCPDLKAVWKIKELPLKKDFCEG (SEQ ID NO: 74) ZN627 MDSVAFEDVAVNFTLEEWALLDPSQKNLYRDVMRETFRNLASVGKQWEDQNIEDPFKIPRRN ISHIPERLCESKEGGQGEETFSQIPDGILNKKTPGVKPCESSVCGEVGMGPSSLNRHIRDHT GREPNEYQEYGKKSYTRNQCGRALSYHRSFPVRERTHPGGKPYDCKECGETFISLVSIRRHM LTHRGGVPYKCKVCGKAFDYPSLFRIHERSHTGEKPYECKQCGKAFSCSSYIRIHERTHTGD KPYECKQCGKAFSCSKYIRIHERTHTGEKPYECKQCGKAFRCASSVRSHERTHTGEKLFECK ECGKALTCLASVRRHMIKHTGNGPYKCKVCGKAFDFPSSFRIHERTHTGEKPYDCKQCGKAF SCSSSFRKHERIHTGEKPYKCTKCGKAFSRSSYFRIHERTHTGEKPYECKQCGKAFSRSTYF RVHEKIHTGEKPYENPNPNASVVPVLS (SEQ ID NO: 666) KAP1 MAASAAAASAAAASAASGSPGPGEGSAGGEKRSTAPSAAASASASAAASSPAGGGAEALELL EHCGVCRERLRPEREPRLLPCLHSACSACLGPAAPAAANSSGDGGAAGDGTVVDCPVCKQQC FSKDIVENYFMRDSGSKAATDAQDANQCCTSCEDNAPATSYCVECSEPLCETCVEAHQRVKY TKDHTVRSTGPAKSRDGERTVYCNVHKHEPLVLFCESCDTLTCRDCQLNAHKDHQYQFLEDA VRNQRKLLASLVKRLGDKHATLQKSTKEVRSSIRQVSDVQKRVQVDVKMAILQIMKELNKRG RVLVNDAQKVTEGQQERLERQHWTMTKIQKHQEHILRFASWALESDNNTALLLSKKLIYFQL HRALKMIVDPVEPHGEMKFQWDLNAWTKSAEAFGKIVAERPGTNSTGPAPMAPPRAPGPLSK QGSGSSQPMEVQEGYGFGSGDDPYSSAEPHVSGVKRSRSGEGEVSGLMRKVPRVSLERLDLD LTADSQPPVFKVFPGSTTEDYNLIVIERGAAAAATGQPGTAPAGTPGAPPLAGMAIVKEEET EAAIGAPPTATEGPETKPVLMALAEGPGAEGPRLASPSGSTSSGLEVVAPEGTSAPGGGPGT LDDSATICRVCQKPGDLVMCNQCEFCFHLDCHLPALQDVPGEEWSCSLCHVLPDLKEEDGSL SLDGADSTGVVAKLSPANQRKCERVLLALFCHEPCRPLHQLATDSTFSLDQPGGTLDLTLIR ARLQEKLSPPYSSPQEFAQDVGRMFKQFNKLTEDKADVQSIIGLQRFFETRMNEAFGDTKFS AVLVEPPPMSLPGAGLSSQELSGGPGDGP (SEQ ID NO: 629) MeCP2 MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKA ETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYL INPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGR GRGRPKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMPFQTSPGGKAEGGGATTSTQVMVI KRPGRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHHHHH SESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPA KTQPAVATAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS (SEQ ID NO: 630) HP1b MGKKQNKKKVEEVLEEEEEEYVVEKVLDRRVVKGKVEYLLKWKGFSDEDNTWEPEENLDCPD LIAEFLQSQKTAHETDKSEGGKRKADSDSEDKGEESKPKKKKEESEKPRGFARGLEPERIIG ATDSSGELMFLMKWKNSDEADLVPAKEANVKCPQVVISFYEERLTWHSYPSEDDDKKDDKN (SEQ ID NO: 94) CBX8 GSGPPSSGGGLYRDMGAQGGRPSLIARIPVARILGDPEEESWSPSLTNLEKVVVTDVTSNFL TVTIKESNTDQGFFKEKR (SEQ ID NO: 387) CDYL2 ASGDLYEVERIVDKRKNKKGKWEYLIRWKGYGSTEDTWEPEHHLLHCEEFIDEFNGLHMSKD KRIKSGKQSSTSKLLRDS (SEQ ID NO: 365) TOX KDPNEPQKPVSAYALFFRDTQAAIKGQNPNATFGEVSKIVASMWDGLGEEQKQVYKKKTEAA KKEYLKQLAAYRASLVSK (SEQ ID NO: 372) TOX3 KDPNEPQKPVSAYALFFRDTQAAIKGQNPNATFGEVSKIVASMWDSLGEEQKQVYKRKTEAA KKEYLKALAAYRASLVSK (SEQ ID NO: 394) TOX4 KDPNEPQKPVSAYALFFRDTQAAIKGQNPNATFGEVSKIVASMWDSLGEEQKQVYKRKTEAA KKEYLKALAAYKDNQECQ (SEQ ID NO: 1529) EED MSEREVSTAPAGTDMPAAKKQKLSSDENSNPDLSGDENDDAVSIESGTNTERPDTPTNTPNA PGRKSWGKGKWKSKKCKYSFKCVNSLKEDHNQPLFGVQFNWHSKEGDPLVFATVGSNRVTLY ECHSQGEIRLLQSYVDADADENFYTCAWTYDSNTSHPLLAVAGSRGIIRIINPITMQCIKHY VGHGNAINELKFHPRDPNLLLSVSKDHALRLWNIQTDTLVAIFGGVEGHRDEVLSADYDLLG EKIMSCGMDHSLKLWRINSKRMMNAIKESYDYNPNKTNRPFISQKIHFPDFSTRDIHRNYVD CVRWLGDLILSKSCENAIVCWKPGKMEDDIDKIKPSESNVTILGRFDYSQCDIWYMRESMDF WQKMLALGNQVGKLYVWDLEVEDPHKAKCTTLTHHKCGAAIRQTSFSRDSSILIAVCDDASI WRWDRLR (SEQ ID NO: 563) RBBP4 VWDLSKIGEEQSPEDAEDGPPELLFIHGGHTAKISDFSWNPNEPWVICSVSEDNIMQVWQMA ENIYNDEDPEGSVDPEGQ (SEQ ID NO: 1530) RCOR1 MPAMVEKGPEVSGKRRGRNNAAASASAAAASAAASAACASPAATAASGAAASSASAAAASAA AAPNNGQNKSLAAAAPNGNSSSNSWEEGSSGSSSDEEHGGGGMRVGPQYQAVVPDFDPAKLA RRSQERDNLGMLVWSPNQNLSEAKLDEYIAIAKEKHGYNMEQALGMLFWHKHNIEKSLADLP NFTPFPDEWTVEDKVLFEQAFSFHGKTFHRIQQMLPDKSIASLVKFYYSWKKTRTKTSVMDR HARKQKREREESEDELEEANGNNPIDIEVDQNKESKKEVPPTETVPQVKKEKHSTQAKNRAK RKPPKGMFLSQEDVEAVSANATAATTVLRQLDMELVSVKRQIQNIKQTNSALKEKLDGGIEP YRLPEVIQKCNARWTTEEQLLAVQAIRKYGRDFQAISDVIGNKSVVQVKNFFVNYRRRENID EVLQEWEAEHGKEETNGPSNQKPVKSPDNSIKMPEEEDEAPVLDVRYASAS (SEQ ID NO: 559) SCML2 KQGFSKDPSTWSVDEVIQFMKHTDPQISGPLADLFRQHEIDGKALFLLKSDVMMKYMGLKLG PALKLCYYIEKLKEGKYS (SEQ ID NO: 382)

Example 15: ZIM 3 Fusion Constructs

Novel fusions of ZIM3 and KOX1KRAB are generated. Both ZIM 3 and KOX1KRAB are KRAB family proteins with extensive homology. Thus, sequences are designed which represent halfway points between ZIM3 and KOX1KRAB. These KOX1KRAB and ZIM3 constructs encode a small region of KOX1KRAB and ZIM3 focused around the zinc finger domain of the protein. While the regions used of KOX1KRAB and ZIM3 are very similar within the first ~75 bp of their sequence, ZIM3 also possesses a small alpha-helical region at the C-terminus, not present in KOX1KRAB. The KOX1KRAB-FL sequence includes the KOX1KRAB sequence equivalent of this extra piece, while the ZIM3 truncation has this extra piece removed from the ZIM3 sequence. The ZIM3/KOX1KRAB chimeras are fusions of the N- and C-terminal pieces of the two proteins. The ZIM3-like KOX1KRAB variants were both assembled by first, BLAST of ZIM3 or KOX1KRAB proteins from nonhuman species to assemble the closest 100 homologs (‘families’) of each gene; second, identifying the 3 members of the KOX1KRAB family that most closely resemble ZIM3 and the 3 members of the ZIM3 family that most closely resemble KOX1KRAB; and third, rationally modifying the KOX1KRAB-FL sequence to resemble each set of three (Table 15).

TABLE 15 ZIM-KOX1KRAB Chimera Proteins Description Sequence KOX1KRAB Residues 11-72 58 KOX1KRAB-FL Residues 11-108 59 ZIM3 Residues 1-100 60 KOX1KRAB-ZIM3 chimera 1 61 (Residues 11-72 of KOX1KRAB, Residues 68-100 of ZIM3) ZIM3-KOX1KRAB chimera 2 62 (Residues 1-67 of ZIM3, Residues 73-108 of KOX1KRAB) Truncated ZIM3 (Residues 1-75 of ZIM3) 63 KOX1KRAB-FL Zim3-like (Modified Residues 11-108) 64 KOX1KRAB-like ZIM3 1 65 KOX1KRAB-like ZIM3 2 66 KOX1KRAB-like ZIM3 3 67 KOX1KRAB-like ZIM3 4 68 ZIM-like KOX1KRAB 1 69 ZIM-like KOX1KRAB 2 70 ZIM-like KOX1KRAB 3 71

Sequences

The SEQ ID NOs (SEQ) of nucleotide (nt) and amino acid (aa) sequences described in the present disclosure are listed below.

SEQ Description Sequence 1 S. pyogenes WT ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGC Cas9 Sequence (nt) GTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAA AAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAA AATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAA GCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGG AAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATG GCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTT TTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGA AATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATC TATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGAT TTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGT GGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGAT GTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTA TTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCG ATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTC ATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAAT CTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAAT TTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACT TACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAA TATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATT TTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCT CCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAA GACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAA AAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCA GGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTT ATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTG GTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTT GACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCAT GCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGAC AATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTAT TATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATG ACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAA GTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATG ACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAA CATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACA AAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTT TCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACA AATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAA AAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGAT AGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATT ATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATC TTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAG ATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGAT AAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGA CGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCT GGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAAT CGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAA GAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTA CATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAA GGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTA ATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGT GAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGT ATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATT CTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAG CTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGAC CAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCAC ATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAG GTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTT CCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAA CTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTA ACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGT TTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCAT GTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAA AATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCT AAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTA CGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAAT GCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAA TCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAA ATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAA TATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATT ACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACT AATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTT GCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTC AAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATT TTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGAC TGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCT TATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAG AAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAA AGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAA GGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAA TATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCT AGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGC AAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTG AAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAG CAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAA TTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTT CTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAA GCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCT CCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGA TATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAA TCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTA GGAGGTGACTGA 2 S. pyogenes WT MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK Cas9 Sequence (aa) NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNEDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDELKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDE ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ AENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD 3 SaCas9 MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYE ARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTK EQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEA KQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKD IKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRD ENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRV TSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILD ELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQ KRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEA IPLEDLLNNPENYEVDHIIPRSVSEDNSENNKVLVKQEENSKKGN RTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEER DINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS INGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKD FKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYD KDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLY KYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSK CYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDIL GNLYEVKSKKHPQIIKKG 4 F. novicida WT MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAK Cpf1 DYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSD DDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLENQNLIDAKKGQE SDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFK GEHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKA PEAINYEQIKKDLAEELTFDIDYKTSEVNQRVESLDEVFEIANEN NYLNQSGITKENTIIGGKFVNGENTKRKGINEYINLYSQQINDKT LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIA AFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQ VFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQN KDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKL KIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNY ITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFS AKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKE IDFYKQSISKHPEWKDFGFRESDTQRYNSIDEFYREVENQGYKLT FENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKA LFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKD NPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKENDEI NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTENIIG NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVH EIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKL NYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAG FTSKICPVTGFVNQLYPKYESVSKSQEFFSKEDKICYNLDKGYFE FSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYP TKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTIL QMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAY HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 5 CasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEK RRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDD HVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPL FVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEA VTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVG KALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGK ENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLS RDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDM GRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKR QFGDLLLYLEKKYAGDWGKVEDEAWERIDKKIAGLTSHIEREEAR NAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWY GDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENG KREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGKAKVIDL TFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVI EKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVDRGE NIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQA AKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAV LVFENLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTY LSKTLAQYTSKTCSNCGFTITTADYDGMLVRLKKTSDGWATTLNN KELKAEGQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTK GRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHADEQAALNIARS WLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA 6 CasY MRKKLFKGYILHNKRLVYTGKAAIRSIKYPLVAPNKTALNNLSEK IIYDYEHLFGPLNVASYARNSNRYSLVDFWIDSLRAGVIWQSKST SLIDLISKLEGSKSPSEKIFEQIDFELKNKLDKEQFKDIILLNTG IRSSSNVRSLRGRFLKCFKEEFRDTEEVIACVDKWSKDLIVEGKS ILVSKQFLYWEEEFGIKIFPHFKDNHDLPKLTFFVEPSLEFSPHL PLANCLERLKKEDISRESLLGLDNNESAFSNYENELENLLSRGEI KKIVTAVLAVSKSWENEPELEKRLHELSEKAKLLGYPKLTSSWAD YRMIIGGKIKSWHSNYTEQLIKVREDLKKHQIALDKLQEDLKKVV DSSLREQIEAQREALLPLLDTMLKEKDESDDLELYRFILSDEKSL LNGSYQRYIQTEEERKEDRDVTKKYKDLYSNLRNIPREFGESKKE QFNKFINKSLPTIDVGLKILEDIRNALETVSVRKPPSITEEYVTK QLEKLSRKYKINAFNSNRFKQITEQVLRKYNNGELPKISEVFYRY PRESHVAIRILPVKISNPRKDISYLLDKYQISPDWKNSNPGEVVD LIEIYKLTLGWLLSCNKDESMDESSYDLKLFPEAASLIKNFGSCL SGYYLSKMIFNCITSEIKGMITLYTRDKFVVRYVTQMIGSNQKEP LLCLVGEKQTKNFSRNWGVLIEEKGDLGEEKNQEKCLIFKDKTDE AKAKEVEIFKNNIWRIRTSKYQIQFLNRLFKKTKEWDLMNLVLSE PSLVLEEEWGVSWDKDKLLPLLKKEKSCEERLYYSLPLNLVPATD YKEQSAEIEQRNTYLGLDVGEFGVAYAVVRIVRDRIELLSWGELK DPALRKIRERVQDMKKKQVMAVFSSSSTAVARVREMAIHSLRNQI HSIALAYKAKIIYEISISNFETGGNRMAKIYRSIKVSDVYRESGA DTLVSEMIWGKKNKQMGNHISSYATSYTCCNCARTPFELVIDNDK EYEKGGDEFIFNVGDEKKVRGFLQKSLLGKTIKGKEVLKSIKEYA RPPIREVLLEGEDVEQLLKRRGNSYIYRCPFCGYKTDADIQAALN IACRGYISDNAKDAVKEGERKLDYILEVRKLWEKNGAVLRSAKFL 7 CasPhi MADTPTLFTQFLRHHLPGQRFRKDILKQAGRILANKGEDATIAFL RGKSEESPPDFQPPVKCPIIACSRPLTEWPIYQASVAIQGYVYGQ SLAEFEASDPGCSKDGLLGWFDKTGVCTDYFSVQGLNLIFQNARK RYIGVQTKVTNRNEKRHKKLKRINAKRIAEGLPELTSDEPESALD ETGHLIDPPGLNTNIYCYQQVSPKPLALSEVNQLPTAYAGYSTSG DDPIQPMVTKDRLSISKGQPGYIPEHQRALLSQKKHRRMRGYGLK ARALLVIVRIQDDWAVIDLRSLLRNAYWRRIVQTKEPSTITKLLK LVTGDPVLDATRMVATFTYKPGIVQVRSAKCLKNKQGSKLESERY LNETVSVTSIDLGSNNLVAVATYRLVNGNTPELLQRFTLPSHLVK DFERYKQAHDTLEDSIQKTAVASLPQGQQTEIRMWSMYGFREAQE RVCQELGLADGSIPWNVMTATSTILTDLFLARGGDPKKCMFTSEP KKKKNSKQVLYKIRDRAWAKMYRTLLSKETREAWNKALWGLKRGS PDYARLSKRKEELARRCVNYTISTAEKRAQCGRTIVALEDLNIGE FHGRGKQEPGWVGLFTRKKENRWLMQALHKAFLELAHHRGYHVIE VNPAYTSQTCPVCRHCDPDNRDQHNREAFHCIGCGFRGNADLDVA THNIAMVAITGESLKRARGSVASKTPQPLAAE 8 Cas12fl (Cas14a) MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEW MGFSSDYKDNHGEYPKSKDILGYTNVHGYAYHTIKTKAYRLNSGN LSQTIKRATDRFKAYQKEILRGDMSIPSYKRDIPLDLIKENISVN RMNHGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRI LSGEYQVSASQIIHDDRKNKWYLNISYDFEPQTRVLDLNKIMGID LGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKY AGGARGGHGRDKRIKPIEQLRDKIANERDTTNHRYSRYIVDMAIK EGCGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKIIYKAEEAGIKV IKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAA RNIAIPNIDKIIAESIKSGGS 9 Cas12f2 (Cas14b) NAMIAQKTIKIKLNPTKEQIIKLNSIIEEYIKVSNFTAKKIAEIQ ESFTDSGLTQGTCSECGKEKTYRKYHLLKKDNKLFCITCYKRKYS QFTLQKVEFQNKTGLRNVAKLPKTYYTNAIRFASDTFSGFDEIIK KKQNRLNSIQNRLNEWKELLYNPSNRNEIKIKVVKYAPKTDTREH PHYYSEAEIKGRIKRLEKQLKKFKMPKYPEFTSETISLQRELYSW KNPDELKISSITDKNESMNYYGKEYLKRYIDLINSQTPQILLEKE NNSFYLCFPITKNIEMPKIDDTFEPVGIDWGITRNIAVVSILDSK TKKPKFVKFYSAGYILGKRKHYKSLRKHFGQKKRQDKINKLGTKE DRFIDSNIHKLAFLIVKEIRNHSNKPIILMENITDNREEAEKSMR QNILLHSVKSRLQNYIAYKALWNNIPTNLVKPEHTSQICNRCGHQ DRENRPKGSKLFKCVKCNYMSNADENASINIARKFYIGEYEPFYK DNEKMKSGVNSISM 10 Cas12f3 (Cas14c) MEVQKTVMKTLSLRILRPLYSQEIEKEIKEEEKERRKQAGGTGEL DGGFYKKLEKKHSEMFSFDRLNLLLNQLQREIAKVYNHAISELYI ATIAQGNKSNKHYISSIVYNRAYGYFYNAYIALGICSKVEANERS NELLTQQSALPTAKSDNFPIVLHKQKGAEGEDGGFRISTEGSDLI FEIPIPFYEYNGENRKEPYKWVKKGGQKPVLKLILSTFRRQRNKG WAKDEGTDAEIRKVTEGKYQVSQIEINRGKKLGEHQKWFANFSIE QPIYERKPNRSIVGGLDVGIRSPLVCAINNSFSRYSVDSNDVEKE SKQVFAFRRRLLSKNSLKRKHGHAAHKLEPITEMTEKNDKERKKI IERWAKEVTNFFVKNQVGIVQIEDLSTMKDREDHFFNQYLRGEWP YYQMQTLIENKLKEYGIEVKRVQAKYTSQLCSNPNCRYWNNYENE EYRKVNKFPKFKCEKCNLEISADYNAARNLSTPDIEKFVAKATKG INLPEK 11 C2c8 MKVLEFKIHPTEEQVSKIDQSLAACKLLWNLSIALKEESKQRYYR KKHKFDEFSPEIWGLSYSGHYDEKEFKTLKDKEKKLLIGNPCCKI AYFKKTSNGKEYTPLNSIPIRREMNAENIDKDAVNYLNRKKLAFY FRENTAKFIGEIETEFKKGFFKSVIKPAYDAAKKGIRGIPREKGR RDKVETLVNGQPETIKIKSNGVIVSSKIGLLKIRGLDRLQGKAPR MAKITRKATGYYLQLTIETDDTIYKESDKCVGLDMGAVAIFTDDL GRQSEAKRYAKIQKKRLNRLQRQASRQKDNSNNQRKTYAKLARVH EKIARQRKGRNAQLAHKITSEYQSVILEDLNLKNMTAAAKPKERE DGDGYKQNGKKRKSGLNKALLDNAIGQLRTFIENKANERGRKIIR VNPKHTSQTCPNCGNIDKANRVSQSKFKCVSCGYEAHADQNAAAN ILIRGLRDEFLRAIGSLYKFPVSMIGKYPGLAGEFTPDLDANQES IGDAPIENAEHSISKQMKQEGNRTPTQPENGSQSLIFLSAPPQPC GDSHGTNNPKALPNKASKRSSKKPRGAIPENPDQLTIWDLLD 12 dSpCas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNEDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTE DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDELKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLEVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ AENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGD 13 dSaCas9 MKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYE ARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTK EQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEA KQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKD IKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRD ENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRV TSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILD ELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQ KRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEA IPLEDLLNNPENYEVDHIIPRSVSEDNSENNKVLVKQEEASKKGN RTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEER DINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS INGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKD FKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYD KDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLY KYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSK CYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDIL GNLYEVKSKKHPQIIKKG 14 inactive FnCpfl MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAK DYKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSD DDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLENQNLIDAKKGQE SDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFK GFHENRKNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKA PEAINYEQIKKDLAEELTFDIDYKTSEVNQRVESLDEVFEIANEN NYLNQSGITKENTIIGGKFVNGENTKRKGINEYINLYSQQINDKT LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIA AFKTVEEKSIKETLSLLEDDLKAQKLDLSKIYFKNDKSLTDLSQQ VFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIEDEIAQN KDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKL KIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNY ITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFS AKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKE IDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLT FENISESYIDSVVNQGKLYLFQIYNKDESAYSKGRPNLHTLYWKA LEDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKD NPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKENDEI NLLLKEKANDVHILSIARGERHLAYYTLVDGKGNIIKQDTENIIG NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVH EIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKL NYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAG FTSKICPVTGFVNQLYPKYESVSKSQEFFSKEDKICYNLDKGYFE FSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYP TKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTIL QMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAY HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 15 dNmeCas9 MAAFKPNSINYILGLAIGIASVGWAMVEIDEEENPIRLIDLGVRV FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREG VLQAANFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLI KHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGDFRTPAEL ALNKFEKESGHIRNQRSDYSHTESRKDLQAELILLFEKQKEFGNP HVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKN TYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKL TYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRAL EKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQP EILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG DHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRY GSPARIHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREY FPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVE IDAALPESRTWDDSENNKVLVLGSENQNKGNQTPYEYENGKDNSR EWQEFKARVETSRFPRSKKQRILLQKEDEDGFKERNLNDTRYVNR FLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAEND RHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEV LHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLL AEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDE GVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNA TMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL IDDSFNFKFSLHPNDLVEVITKKARMEGYFASCHRGTGNINIRIH DLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPP VR 16 dCjCas9 MARILAFAIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLA LPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSEDESL AKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIK NSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKEKENSKE FTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSESKKFEEE VLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTR IINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGL SDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDIT LIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVT PLMLEGKKYDEACNELNLKVAINEDKKDELPAFNETYYKDEVTNP VVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIE KEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYS GEKIKISDLQDEKMLEIDAIYPYSRSFDDSYMNKVLVFTKQNQEK LNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQK NEKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGS KVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNS IVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPESGFRQK VLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLK ALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFA LKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKD MQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEK EVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDEKK 17 dSt1Cas9 MGSDLVLGLAIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNL VRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISININPY QLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGD YAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDETVEKDGKKHR LINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRK YYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAA KASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAM GPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTL ETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGSFSQK QVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTI LTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVN AAIKEYGDEDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAML KAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTIS IHDLINNSNQFEVDAILPLSITEDDSLANKVLVYATANQEKGQRT PYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKED VRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFT SQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSY SEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSIL FSYQVDSKENRKISDATIYATRQAKVGKDKADETYVLGKIKDIYT QDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQIN EKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGN HIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQ FEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDT ETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNV ANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDE 18 dSt3Cas9 MTKPYSIGLAIGTNSVGWAVITDNYKVPSKKMKVLGNTSKKYIKK NLLGVLLFDSGITAEGRRLKRTARRRYTRRRNRILYLQEIFSTEM ATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEEKVYHDEFPTI YHLRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEENSKNND IQKNFQDELDTYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRI LKLFPGEKNSGIFSEFLKLIVGNQADERKCFNLDEKASLHESKES YDEDLETLLGYIGDDYSDVFLKAKKLYDAILLSGELTVTDNETEA PLSSAMIKRYNEHKEDLALLKEYIRNISLKTYNEVEKDDTKNGYA GYIDGKTNQEDFYVYLKNLLAEFEGADYFLEKIDREDELRKQRTF DNGSIPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTFRIPY YVGPLARGNSDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINRM TSFDLYLPEEKVLPKHSLLYETENVYNELTKVRFIAESMRDYQFL DSKQKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDGIELKGIEKQ FNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREM IKQRLSKFENIFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSG NTILDYLIDDGISNRNEMQLIHDDALSFKKKIQKAQIIGDEDKGN IKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVEMA RENQYTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDN NALQNDRLYLYYLQNGKDMYTGDDLDIDRLSNYDIDHIIPQAFLK DNSIDNKVLVSSASARGKSDDFPSLEVVKKRKTFWYQLLKSKLIS QRKFDNLTKAERGGLLPEDKAGFIQRQLVETRQITKHVARLLDEK FNNKKDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREINDEHH AHDAYLNAVIASALLKKYPKLEPEFVYGDYPKYNSFRERKSATEK VYFYSNIMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESDL ATVRRVLSYPQVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPN SNENLVGAKEYLDPKKYGGYAGISNSFAVLVKGTIEKGAKKKITN VLEFQGISILDRINYRKDKLNELLEKGYKDIELIIELPKYSLFEL SDGSRRMLASILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRISN TINENHRKYVENHKKEFEELFYYILEFNENYVGAKKNGKLLNSAF QSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFEFLGVKI PRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKLGEG 19 dLbCpf1 MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLERKKTRTEKE NKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDK DEIALVNSENGFTTAFTGFFDNRENMESEEAKSTSIAFRCINENL TRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFF NFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKL PKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVERNTLNKNSEIF SSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRD KWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYAD ADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKND AVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYD ILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKET DYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKL LPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMENLN DCHKLIDFFKDSISRYPKWSNAYDENFSETEKYKDIAGFYREVEE QGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLH TMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPI ANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIF KINTEVRVLLKHDDNPYVIGIARGERNLLYIVVVDGKGNIVEQYS LNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELK AGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQK FEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQN GFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSEDRIM YVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKK NNVFDWEEVCLTSAYKELENKYGINYQQGDIRALLCEQSDKAFYS SFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQ ENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISN KEWLEYAQTSVKH 20 inactive AsCpf1 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEE TRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLEKA ELFNGKVLKQLGTVTTTEHENALLRSEDKETTYFSGFYENRKNVF SAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVESFPFYNQLLTQTQIDLYNQLLGGISREAG TEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLEKQILSDRNT LSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSA KEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAAL DQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPE FSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL ASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSN NFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDELSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYH ISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYW TGLESPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKK LKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVS HEIIKDRRFTSDKFFFHVPITLNYQAANSPSKENQRVNAYLKEHP ETPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLD NREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEK VGGVLNPYQLTDQFTSFAKMGTQSGELFYVPAPYTSKIDPLTGFV DPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSF QRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVERDGSNILPKLLENDDSHAIDTM VALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM DADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQEL RN 21 inactive enAsCpf1 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEE TRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKA ELFNGKVLKQLGTVTTTEHENALLRSEDKFTTYFSGFYRNRKNVE SAEDISTAIPHRIVQDNEPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVESFPFYNQLLTQTQIDLYNQLLGGISREAG TEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLEKQILSDRNT LSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALENELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSA KEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAAL DQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPE FSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL ARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSN NFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDELSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYH ISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYW TGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKK LKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVS HEIIKDRRFTSDKFFFHVPITLNYQAANSPSKENQRVNAYLKEHP ETPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLD NREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEK VGGVLNPYQLTDQFTSFAKMGTQSGELFYVPAPYTSKIDPLTGFV DPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSF QRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVERDGSNILPKLLENDDSHAIDTM VALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM DADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQEL RN 22 inactive HFAsCpf1 MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEE TRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKA ELFNGKVLKQLGTVTTTEHENALLRSEDKFTTYFSGFYRNRKNVE SAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVESFPFYNQLLTQTQIDLYNQLLGGISREAG TEKIKGLNEVLALAIQKNDETAHIIASLPHRFIPLFKQILSDRNT LSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSA KEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAAL DQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPE FSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL ARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSN NFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYH ISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYW TGLESPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKK LKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVS HEIIKDRRFTSDKFFFHVPITLNYQAANSPSKENQRVNAYLKEHP ETPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLD NREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEK VGGVLNPYQLTDQFTSFAKMGTQSGELFYVPAPYTSKIDPLTGFV DPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSF QRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVERDGSNILPKLLENDDSHAIDTM VALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM DADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQEL RN 23 inactive MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND RVRAsCpf1 HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEE TRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKA ELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVE SAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVESFPFYNQLLTQTQIDLYNQLLGGISREAG TEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNT LSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALENELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSA KEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAAL DQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPE FSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL ARGWDVNVEKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSN NFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDELSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYH ISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYW TGLESPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKK LKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVS HEIIKDRRFTSDKFFFHVPITLNYQAANSPSKENQRVNAYLKEHP ETPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLD NREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEK VGGVLNPYQLTDQFTSFAKMGTQSGELFYVPAPYTSKIDPLTGFV DPFVWKTIKNHESRKHFLEGEDELHYDVKTGDFILHFKMNRNLSE QRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVERDGSNILPKLLENDDSHAIDTM VALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM DADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQEL RN 24 inactive MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARND RRAsCpf1 HYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEE TRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKA ELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVF SAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENV KKAIGIFVSTSIEEVESFPFYNQLLTQTQIDLYNQLLGGISREAG TEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNT LSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSA KEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAAL DQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPE FSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTL ARGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPRCSTQLKAVTAHFQTHTTPILLSN NFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDELSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYH ISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYW TGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKK LKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVS HEIIKDRRFTSDKFFFHVPITLNYQAANSPSKENQRVNAYLKEHP ETPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLD NREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVV VLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEK VGGVLNPYQLTDQFTSFAKMGTQSGELFYVPAPYTSKIDPLTGFV DPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSF QRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVERDGSNILPKLLENDDSHAIDTM VALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM DADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQEL RN 25 dCasX MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEK RRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDD HVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPL FVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEA VTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVG KALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGK ENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLS RDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDM GRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKR QFGDLLLYLEKKYAGDWGKVEDEAWERIDKKIAGLTSHIEREEAR NAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWY GDLRGNPFAVEAENRVVDISGESIGSDGHSIQYRNLLAWKYLENG KREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGKAKVIDL TFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVI EKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGE NIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQA AKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAV LVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTY LSKTLAQYTSKTCSNCGFTITTADYDGMLVRLKKTSDGWATTLNN KELKAEGQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTK GRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARS WLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA 26 dCasPhi MPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVA YLQGKSEEEPPNFQPPAKCHVVTKSRDFAEWPIMKASEAIQRYIY ALSTTERAACKPGKSSESHAAWFAATGVSNHGYSHVQGLNLIFDH TLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEV ATNETGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYAGYV RDPNAPIPLGVVRNRCDIQKGCPGYIPEWQREAGTAISPKTGKAV TVPGLSPKKNKRMRRYWRSEKEKAQDALLVTVRIGTDWVVIDVRG LLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDA CGTYARKWTLKGKQTKATLDKLTATQTVALVAIALGQTNPISAGI SRVTQENGALQCEPLDRFTLPDDLLKDISAYRIAWDRNEEELRAR SVEALPEAQQAEVRALDGVSKETARTQLCADFGLDPKRLPWDKMS SNTTFISEALLSNSVSRDQVFFTPAPKKGAKKKAPVEVMRKDRTW ARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSI NYVIEKTRRRTQCQIVIPVIEDLNVRFFHGSGKRLPGWDNFFTAK KENRWFIQGLHKAFSDLRTHRSFYVFEVRPERTSITCPKCGHCEV GNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKREEPR DAQGTAPARKTKKASKSKAPPAEREDQTPAQEPSQTS 27 inactive VRER MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK SpCas9 NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNEKSNEDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTE DNGSIPHQIHLGELHAILRRQEDFYPELKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RENASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDE ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLEVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ AENIIHLFTLTNLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQ SITGLYETRIDLSQLGGD 28 inactive EQR MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK SpCas9 NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNEKSNEDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTE DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDE ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFESPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLEVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ AENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQ SITGLYETRIDLSQLGGD 29 inactive VQR MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK SpCas9 NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNEKSNEDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTE DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLEKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLEVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ AENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQ SITGLYETRIDLSQLGGD 30 inactive SPG MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK SpCas9 NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNEDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTE DNGSIPHQIHLGELHAILRRQEDFYPELKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLEKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDELKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDE ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD WDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDELEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLEVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ AENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQ SITGLYETRIDLSQLGGD 31 inactive SpRY Cas9 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNEDLAEDAKLQLSKDT YDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTE DNGSIPHQIHLGELHAILRRQEDFYPELKDNREKIEKILTFRIPY YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL SGEQKKAIVDLLEKTNRKVTVKQLKEDYFKKIECFDSVEISGVED RENASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS GKTILDELKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEK LYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNK VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE NDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF ATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLIARKKD WDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDELEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLA SAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLEVE QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ AENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQ SITGLYETRIDLSQLGGD 32 inactive KKH MKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE dSaCas9 GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYE ARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTK EQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEA KQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKD IKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRD ENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRV TSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILD ELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQ KRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEA IPLEDLLNNPFNYEVDHIIPRSVSEDNSENNKVLVKQEEASKKGN RTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEER DINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS INGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKD FKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYD KDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLY KYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSK CYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDIL GNLYEVKSKKHPQIIKKG 33 ZIM3 MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLV SVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEKNGDIGGQI WKPKDVKESL 34 ZNF436 MAATLLMAGSQAPVTFEDMAMYLTREEWRPLDAAQRDLYRDVMQE NYGNVVSLDFEIRSENEVNPKQEISEDVQFGTTSERPAENAEENP ESEEGFESGDRSERQW 35 ZNF257 MLENYRNLVFLGIAVSKPDLITCLEQGKEPCNMKRHEMVAKPPVM CSHIAEDLCPERDIKYFFQKVILRRYDKCEHENLQLRKGCKSVDE CKVCK 36 ZNF675 MGLLTFRDVAIEFSLEEWQCLDTAQRNLYKNVILENYRNLVELGI AVSKQDLITCLEQEKEPLTVKRHEMVNEPPVMCSHFAQEFWPEQN IKDSF 37 ZNF490 MLQMQNSEHHGQSIKTQTDSISLEDVAVNFTLEEWALLDPGQRNI YRDVMRATFKNLACIGEKWKDQDIEDEHKNQGRNLRSPMVEALCE NKEDCPCGKSTSQIPDLNTNLETPTG 38 ZNF320 MALSQGLLTFRDVAIEFSQEEWKCLDPAQRTLYRDVMLENYRNLV SLDISSKCMMNTLSSTGQGNTEVIHTGTLQRQASYHIGAFCSQEI EKDIHDFVFQ 39 ZNF331 MAQGLVTFADVAIDFSQEEWACLNSAQRDLYWDVMLENYSNLVSL DLESAYENKSLPTKKNIHEIRASKRNSDRRSKSLGRNWICEGTLE RPQRSRGR 40 ZNF816 MLREEATKKSKEKEPGMALPQGRLTERDVAIEFSLEEWKCLNPAQ RALYRAVMLENYRNLEFVDSSLKSMMEFSSTRHSITGEVIHTGTL QRHKSHHIGDFCFPEMKKDIHHFEFQWQ 41 ZNF680 MPGPPGSLEMGPLTERDVAIEFSLEEWQCLDTAQRNLYRKVMFEN YRNLVFLGIAVSKPHLITCLEQGKEPWNRKRQEMVAKPPVIYSHE TEDLWPEHSIKDSF 42 ZNF41 MSPPWSPALAAEGRGSSCEASVSFEDVTVDFSKEEWQHLDPAQRR LYWDVTLENYSHLLSVGYQIPKSEAAFKLEQGEGPWMLEGEAPHQ SCSGEAIGKMQQQGIPGGIFFHC 43 ZNF189 MASPSPPPESKEEWDYLDPAQRSLYKDVMMENYGNLVSLDVLNRD KDEEPTVKQEIEEIEEEVEPQGVIVTRIKSEIDQDPMGRETFELV GRLDKQRGIFLWEIPRESL 44 ZNF528 MALTQGPLKFMDVAIEFSQEEWKCLDPAQRTLYRDVMLENYRNLV SLGICLPDLSVTSMLEQKRDPWTLQSEEKIANDPDGRECIKGVNT ERSSKLGSN 45 ZNF543 MAASAQVSVTFEDVAVTFTQEEWGQLDAAQRTLYQEVMLETCGLL MSLGCPLFKPELIYQLDHRQELWMATKDLSQSSYPGDNTKPKTTE PTESHLALPE 46 ZNF554 MFSQEERMAAGYLPRWSQELVTFEDVSMDFSQEEWELLEPAQKNL YREVMLENYRNVVSLEALKNQCTDVGIKEGPLSPAQTSQVTSLSS WTGYLLFQPVASSHLEQREALWIEEKGTPQASCSDWMTVLRNQDS TYKKVALQE 47 ZNF140 MSQGSVTFRDVAIDFSQEEWKWLQPAQRDLYRCVMLENYGHLVSL GLSISKPDVVSLLEQGKEPWLGKREVKRDLFSVSESSGEIKDESP KNVIYDD 48 ZNF610 MEEAQKRKAKESGMALPQGRLTEMDVAIEFSQEEWKSLDPGQRAL YRDVMLENYRNLVFLGRSCVLGSNAENKPIKNQLGLTLESHLSEL QLFQAGRKIYRSNQVEKFTNHR 49 ZNF264 MAAAVLTDRAQVSVTFDDVAVTFTKEEWGQLDLAQRTLYQEVMLE NCGLLVSLGCPVPKAELICHLEHGQEPWTRKEDLSQDTCPGDKGK PKTTEPTTCEPALSE 50 ZNF350 MIQAQESITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLV AVGYQASKPDALFKLEQGEQLWTIEDGIHSGACSDIWKVDHVLER LQSESLVNR 51 ZNF8 MEGVAGVMSVGPPAARLQEPVTERDVAVDFTQEEWGQLDPTQRIL YRDVMLETFGHLLSIGPELPKPEVISQLEQGTELWVAERGTTQGC HPAWEPRSESQASRKEEGLPEE 52 ZNF582 MSLGSELFRDVAIVFSQEEWQWLAPAQRDLYRDVMLETYSNLVSL GLAVSKPDVISFLEQGKEPWMVERVVSGGLCPVLESRYDTKELFP KQHVYEV 53 ZNF30 MAHKYVGLQYHGSVTFEDVAIAFSQQEWESLDSSQRGLYRDVMLE NYRNLVSMAGHSRSKPHVIALLEQWKEPEVTVRKDGRRWCTDLQL EDDTIGCKEMPTSEN 54 ZNF324 MAFEDVAVYFSQEEWGLLDTAQRALYRRVMLDNFALVASLGLSTS RPRVVIQLERGEEPWVPSGTDTTLSRTTYRRRNPGSWSLTEDRDV SG 55 ZNF98 MLENYRNLVFVGIAASKPDLITCLEQGKEPWNVKRHEMVTEPPVV YSYFAQDLWPKQGKKNYFQKVILRTYKKCGRENLQLRKYCKSMDE CKVHKECYNGLNQC 56 ZNF669 MHERRPDPCREPLASPIQDSVAFEDVAVNFTQEEWALLDSSQKNL YREVMQETCRNLASVGSQWKDQNIEDHFEKPGKDIRNHIVQRLCE SKEDGQYGEVVSQIPNLDLNENISTGLKPCECSICGK 57 ZNF677 MALSQGLFTFKDVAIEFSQEEWECLDPAQRALYRDVMLENYRNLL SLDEDNIPPEDDISVGFTSKGLSPKENNKEELYHLVILERKESHG INNFDLKEVWENMPKEDSLW 58 ZNF596 MTFEDIIVDFTQEEWALLDTSQRKLFQDVMLENISHLVSIGKQLC KSVVLSQLEQVEKLSTQRISLLQGREVGIKHQEIPFIHHIYQKGT STISTMRS 59 ZNF214 MAVTFEDVTIIFTWEEWKFLDSSQKRLYREVMWENYTNVMSVENW NESYKSQEEKFRYLEYENFSYWQGWWNAGAQMYENQNYGETVQGT DSKDLTQQDRSQC 60 ZNF37A MITSQGSVSFRDVTVGFTQEEWQHLDPAQRTLYRDVMLENYSHLV SVGYCIPKPEVILKLEKGEEPWILEEKFPSQSHLELINTSRNYSI MKFNEENKG 61 ZNF34 MFEDVAVYLSREEWGRLGPAQRGLYRDVMLETYGNLVSLGVGPAG PKPGVISQLERGDEPWVLDVQGTSGKEHLRVNSPALGTRTEYKEL TSQETFGEEDPQGSEPVEACDHIS 62 ZNF250 METYGNVVSLGLPGSKPDIISQLERGEDPWVLDRKGAKKSQGLWS DYSDNLKYDHTTACTQQDSLSCPWECETKGESQNTDLSPKPLISE QTVILGKTPLGRIDQENNETKQ 63 ZNF547 MAEMNPAQGHVVFEDVAIYFSQEEWGHLDEAQRLLYRDVMLENLA LLSSLGCCHGAEDEEAPLEPGVSVGVSQVMAPKPCLSTQNTQPCE TCSSLLKDILRL 64 ZNF273 MLDNYRNLVELGIAVSKPDLITCLEQGKEPCNMKRHAMVAKPPVV CSHFAQDLWPKQGLKDS 65 ZNF354A MAAGQREARPQVSLTFEDVAVLFTRDEWRKLAPSQRNLYRDVMLE NYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLGSKSS HKTTKSTQTQDSSFQ 66 ZFP82 MALRSVMFSDVSIDESPEEWEYLDLEQKDLYRDVMLENYSNLVSL GCFISKPDVISSLEQGKEPWKVVRKGRRQYPDLETKYETKKLSLE NDIYEIN 67 ZNF224 MTTFKEAMTFKDVAVVFTEEELGLLDLAQRKLYRDVMLENERNLL SVGHQAFHRDTFHFLREEKIWMMKTAIQREGNSGDKIQTEMETVS EAGTHQEW 68 ZNF33A MFQVEQKSQESVSFKDVTVGFTQEEWQHLDPSQRALYRDVMLENY SNLVSVGYCVHKPEVIFRLQQGEEPWKQEEEFPSQSFPEVWTADH LKERSQENQSKHL 69 ZNF45 MTKSKEAVTFKDVAVVFSEEELQLLDLAQRKLYRDVMLENERNVV SVGHQSTPDGLPQLEREEKLWMMKMATQRDNSSGAKNLKEMETLQ EVGLRYLP 70 ZNF175 MSQKPQVLGPEKQDGSCEASVSFEDVTVDESREEWQQLDPAQRCL YRDVMLELYSHLFAVGYHIPNPEVIFRMLKEKEPRVEEAEVSHQR CQEREFGLEIPQKEISKKASFQ 71 ZNF595 MELVTFRDVAIEFSPEEWKCLDPAQQNLYRDVMLENYRNLVSLGF VISNPDLVTCLEQIKEPCNLKIHETAAKPPAICSPFSQDLSPVQG IEDSF 72 ZNF184 MSTLLQGGHNLLSSASFQESVTFKDVIVDFTQEEWKQLDPGQRDL FRDVTLENYTHLVSIGLQVSKPDVISQLEQGTEPWIMEPSIPVGT CADWETRLENSVSAPEPDISEE 73 ZNF419 MDPAQVPVAADLLTDHEEGYVTFEDVAVYFSQEEWRLLDDAQRLL YRNVMLENFTLLASLGLASSKTHEITQLESWEEPEMPAWEVVTSA IPRGCWHGAEAEEAPEQIASVG 74 ZFP28-1 MKKLEAVGTGIEPKAMSQGLVTFGDVAVDESQEEWEWLNPIQRNL YRKVMLENYRNLASLGLCVSKPDVISSLEQGKEPWTVKRKMTRAW CPDLKAVWKIKELPLKKDFCEG 75 ZFP28-2 MSLLGEHWDYDALFETQPGLVTIKNLAVDFRQQLHPAQKNECKNG IWENNSDLGSAGHCVAKPDLVSLLEQEKEPWMVKRELTGSLESGQ RSVHETQELFPKQDSYAE 76 ZNF18 MLALAASQPARLEERLIRDRDLGASLLPAAPQEQWRQLDSTQKEQ YWDLILETYGKMVSGAGISHPKSDLTNSIEFGEELAGIYLHVNEK IPRPTCIGDRQENDKENLNLENH 77 ZNF213 MEGRPGETTDTCFVSGVHGPVALGDIPFYESREEWGTLDPAQRDL FWDIKRENSRNTTLGFGLKGQSEKSLLQEMVPVVPGQTGSDVTVS WSPEEAEAWESENRPRAALGPVVGARRGRPPTRRRQFRDLA 78 ZNF394 MVAVVRALQRALDGTSSQGMVTFEDTAVSLTWEEWERLDPARRDE CRESAQKDSGSTVPPSLESRVENKELIPMQQILEEAEPQGQLQEA FQGKRPLESKCGSTHEDRVEKQSGDP 79 ZFP1 MNKSQGSVSFTDVTVDFTQEEWEQLDPSQRILYMDVMLENYSNLL SVEVWKADDQMERDHRNPDEQARQFLILKNQTPIEERGDLFGKAL NLNTDFVSLRQVPYKYDLYEKTL 80 ZFP14 MAHGSVTFRDVAIDESQEEWEFLDPAQRDLYRDVMWENYSNFISL GPSISKPDVITLLDEERKEPGMVVREGTRRYCPDLESRYRTNTLS PEKDIYEIYSFQWDIMER 81 ZNF416 MAAAVLRDSTSVPVTAEAKLMGFTQGCVTFEDVAIYFSQEEWGLL DEAQRLLYRDVMLENFALITALVCWHGMEDEETPEQSVSVEGVPQ VRTPEASPSTQKIQSCDMCVPFLTDILHLTDLPGQELYLTGACAV FHQDQK 82 ZNF557 MLPPTAASQREGHTEGGELVNELLKSWLKGLVTFEDVAVEFTQEE WALLDPAQRTLYRDVMLENCRNLASLGNQVDKPRLISQLEQEDKV MTEERGILSGTCPDVENPFKAKGLTPKLHVERKEQSRNMKMER 83 ZNF566 MAQESVMFSDVSVDFSQEEWECLNDDQRDLYRDVMLENYSNLVSM GHSISKPNVISYLEQGKEPWLADRELTRGQWPVLESRCETKKLFL KKEIYEIESTQWEIMEK 84 ZNF729 MPGAPGSLEMGPLTERDVTIEFSLEEWQCLDTVQQNLYRDVMLEN YRNLVELGMAVEKPDLITCLKQGKEPWNMKRHEMVTKPPVMRSHF TQDLWPDQSTKDSFQEVILRTYAR 85 ZIM2 MAGSQFPDFKHLGTFLVFEELVTFEDVLVDESPEELSSLSAAQRN LYREVMLENYRNLVSLGHQFSKPDIISRLEEEESYAMETDSRHTV ICQGE 86 ZNF254 MPGPPRSLEMGLLTERDVAIEFSLEEWQHLDIAQQNLYRNVMLEN YRNLAFLGIAVSKPDLITCLEQGKEPWNMKRHE 87 ZNF764 MAPPLAPLPPRDPNGAGPEWREPGAVSFADVAVYFCREEWGCLRP AQRALYRDVMRETYGHLSALGIGGNKPALISWVEEEAELWGPAAQ DPE 88 ZNF785 MGPPLAPRPAHVPGEAGPRRTRESRPGAVSFADVAVYESPEEWEC LRPAQRALYRDVMRETFGHLGALGFSVPKPAFISWVEGEVEAWSP EAQDPDGESS 89 ZNF10 (KOX1) MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLE NYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETA FEIKSSVSSRSIFKDKQSCDIKMEGMARNDLWYLSLEEVWKCRDQ LDKYQENPERHLRQVAFTQKKVLTQERVSESGKYGGNCLLPAQLV LREYFHKRDSHTKSLKHDLVLNGHQDSCASNSNECGQTFCQNIHL IQFARTHTGDKSYKCPDNDNSLTHGSSLGISKGIHREKPYECKEC GKFFSWRSNLTRHQLIHTGEKPYECKECGKSFSRSSHLIGHQKTH TGEEPYECKECGKSFSWFSHLVTHQRTHTGDKLYTCNQCGKSFVH SSRLIRHQRTHTGEKPYECPECGKSFRQSTHLILHQRTHVRVRPY ECNECGKSYSQRSHLVVHHRIHTGLKPFECKDCGKCFSRSSHLYS HQRTHTGEKPYECHDCGKSFSQSSALIVHQRIHTGEKPYECCQCG KAFIRKNDLIKHQRIHVGEETYKCNQCGIIFSQNSPFIVHQIAHT GEQFLTCNQCGTALVNTSNLIGYQTNHIRENAY 90 CBX5 MGKKTKRTADSSSSEDEEEYVVEKVLDRRVVKGQVEYLLKWKGES (chromoshadow EEHNTWEPEKNLDCPELISEFMKKYKKMKEGENNKPREKSESNKR domain) KSNESNSADDIKSKKKREQSNDIARGFERGLEPEKIIGATDSCGD LMFLMKWKDTDEADLVLAKEANVKCPQIVIAFYEERLTWHAYPED AENKEKETAKS 91 RYBP MTMGDKKSPTRPKRQAKPAADEGFWDCSVCTERNSAEAFKCSICD (YAF2_RYBP VRKGTSTRKPRINSQLVAQQVAQQYATPPPPKKEKKEKVEKQDKE component of KPEKDKEISPSVTKKNTNKKTKPKSDILKDPPSEANSIQSANATT PRC1) KTSETNHTSRPRLKNVDRSTAQQLAVTVGNVTVIITDFKEKTRSS STSSSTVTSSAGSEQQNQSSSGSESTDKGSSRSSTPKGDMSAVND ESF 92 YAF2 MGDKKSPTRPKRQPKPSSDEGYWDCSVCTFRNSAEAFKCMMCDVR (YAF2 RYBP KGTSTRKPRPVSQLVAQQVTQQFVPPTQSKKEKKDKVEKEKSEKE component of TTSKKNSHKKTRPRLKNVDRSSAQHLEVTVGDLTVIITDFKEKTK PRC1) SPPASSAASADQHSQSGSSSDNTERGMSRSSSPRGEASSLNGESH 93 MGA (component MEEKQQIILANQDGGTVAGAAPTFFVILKQPGNGKTDQGILVINQ of PRC1.6) DACALASSVSSPVKSKGKICLPADCTVGGITVTLDNNSMWNEFYH RSTEMILTKQGRRMFPYCRYWITGLDSNLKYILVMDISPVDNHRY KWNGRWWEPSGKAEPHVLGRVFIHPESPSTGHYWMHQPVSFYKLK LTNNTLDQEGHIILHSMHRYLPRLHLVPAEKAVEVIQLNGPGVHT FTFPQTEFFAVTAYQNIQITQLKIDYNPFAKGERDDGLNNKPQRD GKQKNSSDQEGNNISSSSGHRVRLTEGQGSEIQPGDLDPLSRGHE TSGKGLEKTSLNIKRDELGEMDTDSALSEVPQLKQEISECLIASS FEDDSRVASPLDQNGSENVVIKEEPLDDYDYELGECPEGVTVKQE ETDEETDVYSNSDDDPILEKQLKRHNKVDNPEADHLSSKWLPSSP SGVAKAKMFKLDTGKMPVVYLEPCAVTRSTVKISELPDNMLSTSR KDKSSMLAELEYLPTYIENSNETAFCLGKESENGLRKHSPDLRVV QKYPLLKEPQWKYPDISDSISTERILDDSKDSVGDSLSGKEDLGR KRTTMLKIATAAKVVNANQNASPNVPGKRGRPRKLKLCKAGRPPK NTGKSLISTKNTPVSPGSTFPDVKPDLEDVDGVLFVSFESKEALD IHAVDGTTEESSSLQASTTNDSGYRARISQLEKELIEDLKTLRHK QVIHPGLQEVGLKLNSVDPTMSIDLKYLGVQLPLAPATSFPFWNL TGTNPASPDAGFPFVSRTGKINDFTKIKGWRGKFHSASASRNEGG NSESSLKNRSAFCSDKLDEYLENEGKLMETSMGFSSNAPTSPVVY QLPTKSTSYVRTLDSVLKKQSTISPSTSYSLKPHSVPPVSRKAKS QNRQATFSGRTKSSYKSILPYPVSPKQKYSHVILGDKVTKNSSGI ISENQANNFVVPTLDENIFPKQISLRQAQQQQQQQQGSRPPGLSK SQVKLMDLEDCALWEGKPRTYITEERADVSLTTLLTAQASLKTKP IHTIIRKRAPPCNNDFCRLGCVCSSLALEKRQPAHCRRPDCMEGC TCLKRKVVLVKGGSKTKHFQRKAAHRDPVFYDTLGEEAREEEEGI REEEEQLKEKKKRKKLEYTICETEPEQPVRHYPLWVKVEGEVDPE PVYIPTPSVIEPMKPLLLPQPEVLSPTVKGKLLTGIKSPRSYTPK PNPVIREEDKDPVYLYFESMMTCARVRVYERKKEDQRQPSSSSSP SPSFQQQTSCHSSPENHNNAKEPDSEQQPLKQLTCDLEDDSDKLQ EKSWKSSCNEGESSSTSYMHQRSPGGPTKLIEIISDCNWEEDRNK ILSILSQHINSNMPQSLKVGSFIIELASQRKSRGEKNPPVYSSRV KISMPSCQDQDDMAEKSGSETPDGPLSPGKMEDISPVQTDALDSV RERLHGGKGLPFYAGLSPAGKLVAYKRKPSSSTSGLIQVASNAKV AASRKPRTLLPSTSNSKMASSSGTATNRPGKNLKAFVPAKRPIAA RPSPGGVFTQFVMSKVGALQQKIPGVSTPQTLAGTQKESIRPSPV MVVTPVVSSEPVQVCSPVTAAVTTTTPQVFLENTTAVTPMTAISD VETKETTYSSGATTTGVVEVSETNTSTSVTSTQSTATVNLTKTTG ITTPVASVAFPKSLVASPSTITLPVASTASTSLVVVTAAASSSMV TTPTSSLGSVPIILSGINGSPPVSQRPENAAQIPVATPQVSPNTV KRAGPRLLLIPVQQGSPTLRPVSNTQLQGHRMVLQPVRSPSGMNL FRHPNGQIVQLLPLHQLRGSNTQPNLQPVMERNPGSVMGIRLPAP SKPSETPPSSTSSSAFSVMNPVIQAVGSSSAVNVITQAPSLLSSG ASFVSQAGTLTLRISPPEPQSFASKTGSETKITYSSGGQPVGTAS LIPLQSGSFALLQLPGQKPVPSSILQHVASLQMKRESQNPDQKDE TNSIKREQETKKVLQSEGEAVDPEANVIKQNSGAATSEETLNDSL EDRGDHLDEECLPEEGCATVKPSEHSCITGSHTDQDYKDVNEEYG ARNRKSSKEKVAVLEVRTISEKASNKTVQNLSKVQHQKLGDVKVE QQKGFDNPEENSSEFPVTFKEESKFELSGSKVMEQQSNLQPEAKE KECGDSLEKDRERWRKHLKGPLTRKCVGASQECKKEADEQLIKET KTCQENSDVFQQEQGISDLLGKSGITEDARVLKTECDSWSRISNP SAFSIVPRRAAKSSRGNGHFQGHLLLPGEQIQPKQEKKGGRSSAD FTVLDLEEDDEDDNEKTDDSIDEIVDVVSDYQSEEVDDVEKNNCV EYIEDDEEHVDIETVEELSEEINVAHLKTTAAHTQSFKQPSCTHI SADEKAAERSRKAPPIPLKLKPDYWSDKLQKEAEAFAYYRRTHTA NERRRRGEMRDLFEKLKITLGLLHSSKVSKSLILTRAFSEIQGLT DQADKLIGQKNLLTRKRNILIRKVSSLSGKTEEVVLKKLEYIYAK QQALEAQKRKKKMGSDEFDISPRISKQQEGSSASSVDLGQMFINN RRGKPLILSRKKDQATENTSPLNTPHTSANLVMTPQGQLLTLKGP LFSGPVVAVSPDLLESDLKPQVAGSAVALPENDDLEMMPRIVNVT SLATEGGLVDMGGSKYPHEVPDSKPSDHLKDTVRNEDNSLEDKGR ISSRGNRDGRVTLGPTQVFLANKDSGYPQIVDVSNMQKAQEFLPK KISGDMRGIQYKWKESESRGERVKSKDSSFHKLKMKDLKDSSIEM ELRKVTSAIEEAALDSSELLTNMEDEDDTDETLTSLLNEIAFLNQ QLNDDSVGLAELPSSMDTEFPGDARRAFISKVPPGSRATFQVEHL GTGLKELPDVQGESDSISPLLLHLEDDDESENEKQLAEPASEPDV LKIVIDSEIKDSLLSNKKAIDGGKNTSGLPAEPESVSSPPTLHMK TGLENSNSTDTLWRPMPKLAPLGLKVANPSSDADGQSLKVMPCLA PIAAKVGSVGHKMNLTGNDQEGRESKVMPTLAPVVAKLGNSGASP SSAGK 94 CBX1 MGKKQNKKKVEEVLEEEEEEYVVEKVLDRRVVKGKVEYLLKWKGF (chromoshadow) SDEDNTWEPEENLDCPDLIAEFLQSQKTAHETDKSEGGKRKADSD SEDKGEESKPKKKKEESEKPRGFARGLEPERIIGATDSSGELMEL MKWKNSDEADLVPAKEANVKCPQVVISFYEERLTWHSYPSEDDDK KDDKN 95 SCMH1 MLVCYSVLACEILWDLPCSIMGSPLGHFTWDKYLKETCSVPAPVH (SAM_1/SPM) CFKQSYTPPSNEFKISMKLEAQDPRNTTSTCIATVVGLTGARLRL RLDGSDNKNDFWRLVDSAEIQPIGNCEKNGGMLQPPLGERLNASS WPMFLLKTLNGAEMAPIRIFHKEPPSPSHNFFKMGMKLEAVDRKN PHFICPATIGEVRGSEVLVTEDGWRGAFDYWCREDSRDIFPVGWC SLTGDNLQPPGTKVVIPKNPYPASDVNTEKPSIHSSTKTVLEHQP GQRGRKPGKKRGRTPKTLISHPISAPSKTAEPLKFPKKRGPKPGS KRKPRTLLNPPPASPTTSTPEPDTSTVPQDAATIPSSAMQAPTVC IYLNKNGSTGPHLDKKKVQQLPDHFGPARASVVLQQAVQACIDCA YHQKTVFSFLKQGHGGEVISAVFDREQHTLNLPAVNSITYVLREL EKLCHNLRSDNLFGNQPFTQTHLSLTAIEYSHSHDRYLPGETFVL GNSLARSLEPHSDSMDSASNPTNLVSTSQRHRPLLSSCGLPPSTA SAVRRLCSRGVLKGSNERRDMESFWKLNRSPGSDRYLESRDASRL SGRDPSSWTVEDVMQFVREADPQLGPHADLERKHEIDGKALLLLR SDMMMKYMGLKLGPALKLSYHIDRLKQGKF 96 MPP8 MEQVAEGARVTAVPVSAADSTEELAEVEEGVGVVGEDNDAAARGA (Chromodomain) EAFGDSEEDGEDVFEVEKILDMKTEGGKVLYKVRWKGYTSDDDTW EPEIHLEDCKEVLLEFRKKIAENKAKAVRKDIQRLSLNNDIFEAN SDSDQQSETKEDTSPKKKKKKLRQREEKSPDDLKKKKAKAGKLKD KSKPDLESSLESLVEDLRTKKRISEAKEELKESKKPKKDEVKETK ELKKVKKGEIRDLKTKTREDPKENRKTKKEKFVESQVESESSVLN DSPFPEDDSEGLHSDSREEKQNTKSARERAGQDMGLEHGFEKPLD SAMSAEEDTDVRGRRKKKTPRKAEDTRENRKLENKNAFLEKKTVP KKQRNQDRSKSAAELEKLMPVSAQTPKGRRLSGEERGLWSTDSAE EDKETKRNESKEKYQKRHDSDKEEKGRKEPKGLKTLKEIRNAFDL FKLTPEEKNDVSENNRKREEIPLDEKTIDDHKTKENKQSLKERRN TRDETDTWAYIAAEGDQEVLDSVCQADENSDGRQQILSLGMDLQL EWMKLEDFQKHLDGKDENFAATDAIPSNVLRDAVKNGDYITVKVA LNSNEEYNLDQEDSSGMTLVMLAAAGGQDDLLRLLITKGAKVNGR QKNGTTALIHAAEKNELTTVAILLEAGAFVNVQQSNGETALMKAC KRGNSDIVRLVIECGADCNILSKHQNSALHFAKQSNNVLVYDLLK NHLETLSRVAEETIKDYFEARLALLEPVFPIACHRLCEGPDESTD FNYKPPQNIPEGSGILLFIFHANFLGKEVIARLCGPCSVQAVVLN DKFQLPVELDSHFVYSFSPVAGPNKLFIRLTEAPSAKVKLLIGAY RVQLQ 97 SUMO3 (Rad60- MSEEKPKEGVKTENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKA SLD) YCERQGLSMRQIRFREDGQPINETDTPAQLEMEDEDTIDVEQQQT GGVPESSLAGHSF 98 HERC2 (Cyt-b5) MPSESFCLAAQARLDSKWLKTDIQLAFTRDGLCGLWNEMVKDGEI VYTGTESTQNGELPPRKDDSVEPSGTKKEDLNDKEKKDEEETPAP IYRAKSILDSWVWGKQPDVNELKECLSVLVKEQQALAVQSATTTL SALRLKQRLVILERYFIALNRTVFQENVKVKWKSSGISLPPVDKK SSRPAGKGVEGLARVGSRAALSFAFAFLRRAWRSGEDADLCSELL QESLDALRALPEASLEDESTVSSVWLEVVERATRFLRSVVTGDVH GTPATKGPGSIPLQDQHLALAILLELAVQRGTLSQMLSAILLLLQ LWDSGAQETDNERSAQGTSAPLLPLLQRFQSIICRKDAPHSEGDM HLLSGPLSPNESFLRYLTLPQDNELAIDLRQTAVVVMAHLDRLAT PCMPPLCSSPTSHKGSLQEVIGWGLIGWKYYANVIGPIQCEGLAN LGVTQIACAEKRFLILSRNGRVYTQAYNSDTLAPQLVQGLASRNI VKIAAHSDGHHYLALAATGEVYSWGCGDGGRLGHGDTVPLEEPKV ISAFSGKQAGKHVVHIACGSTYSAAITAEGELYTWGRGNYGRLGH GSSEDEAIPMLVAGLKGLKVIDVACGSGDAQTLAVTENGQVWSWG DGDYGKLGRGGSDGCKTPKLIEKLQDLDVVKVRCGSQFSIALTKD GQVYSWGKGDNQRLGHGTEEHVRYPKLLEGLQGKKVIDVAAGSTH CLALTEDSEVHSWGSNDQCQHFDTLRVTKPEPAALPGLDTKHIVG IACGPAQSFAWSSCSEWSIGLRVPFVVDICSMTFEQLDLLLRQVS EGMDGSADWPPPQEKECVAVATLNLLRLQLHAAISHQVDPEFLGL GLGSILLNSLKQTVVTLASSAGVLSTVQSAAQAVLQSGWSVLLPT AEERARALSALLPCAVSGNEVNISPGRREMIDLLVGSLMADGGLE SALHAAITAEIQDIEAKKEAQKEKEIDEQEANASTFHRSRTPLDK DLINTGICESSGKQCLPLVQLIQQLLRNIASQTVARLKDVARRIS SCLDFEQHSRERSASLDLLLRFQRLLISKLYPGESIGQTSDISSP ELMGVGSLLKKYTALLCTHIGDILPVAASIASTSWRHFAEVAYIV EGDFTGVLLPELVVSIVLLLSKNAGLMQEAGAVPLLGGLLEHLDR FNHLAPGKERDDHEELAWPGIMESFFTGQNCRNNEEVTLIRKADL ENHNKDGGFWTVIDGKVYDIKDFQTQSLTGNSILAQFAGEDPVVA LEAALQFEDTRESMHAFCVGQYLEPDQEIVTIPDLGSLSSPLIDT ERNLGLLLGLHASYLAMSTPLSPVEIECAKWLQSSIFSGGLQTSQ IHYSYNEEKDEDHCSSPGGTPASKSRLCSHRRALGDHSQAFLQAI ADNNIQDHNVKDFLCQIERYCRQCHLTTPIMFPPEHPVEEVGRLL LCCLLKHEDLGHVALSLVHAGALGIEQVKHRTLPKSVVDVCRVVY QAKCSLIKTHQEQGRSYKEVCAPVIERLRFLFNELRPAVCNDLSI MSKFKLLSSLPRWRRIAQKIIRERRKKRVPKKPESTDDEEKIGNE ESDLEEACILPHSPINVDKRPIAIKSPKDKWQPLLSTVTGVHKYK WLKQNVQGLYPQSPLLSTIAEFALKEEPVDVEKMRKCLLKQLERA EVRLEGIDTILKLASKNFLLPSVQYAMFCGWQRLIPEGIDIGEPL TDCLKDVDLIPPENRMLLEVTFGKLYAWAVQNIRNVLMDASAKFK ELGIQPVPLQTITNENPSGPSLGTIPQARFLLVMLSMLTLQHGAN NLDLLLNSGMLALTQTALRLIGPSCDNVEEDMNASAQGASATVLE ETRKETAPVQLPVSGPELAAMMKIGTRVMRGVDWKWGDQDGPPPG LGRVIGELGEDGWIRVQWDTGSTNSYRMGKEGKYDLKLAELPAAA QPSAEDSDTEDDSEAEQTERNIHPTAMMFTSTINLLQTLCLSAGV HAEIMQSEATKTLCGLLRMLVESGTTDKTSSPNRLVYREQHRSWC TLGFVRSIALTPQVCGALSSPQWITLLMKVVEGHAPFTATSLQRQ ILAVHLLQAVLPSWDKTERARDMKCLVEKLFDFLGSLLTTCSSDV PLLRESTLRRRRVRPQASLTATHSSTLAEEVVALLRTLHSLTQWN GLINKYINSQLRSITHSFVGRPSEGAQLEDYFPDSENPEVGGLMA VLAVIGGIDGRLRLGGQVMHDEFGEGTVTRITPKGKITVQFSDMR TCRVCPLNQLKPLPAVAFNVNNLPFTEPMLSVWAQLVNLAGSKLE KHKIKKSTKQAFAGQVDLDLLRCQQLKLYILKAGRALLSHQDKLR QILSQPAVQETGTVHTDDGAVVSPDLGDMSPEGPQPPMILLQQLL ASATQPSPVKAIFDKQELEAAALAVCQCLAVESTHPSSPGFEDCS SSEATTPVAVQHIRPARVKRRKQSPVPALPIVVQLMEMGESRRNI EFALKSLTGASGNASSLPGVEALVGWLLDHSDIQVTELSDADTVS DEYSDEEVVEDVDDAAYSMSTGAVVTESQTYKKRADFLSNDDYAV YVRENIQVGMMVRCCRAYEEVCEGDVGKVIKLDRDGLHDLNVQCD WQQKGGTYWVRYIHVELIGYPPPSSSSHIKIGDKVRVKASVTTPK YKWGSVTHQSVGVVKAFSANGKDIIVDFPQQSHWTGLLSEMELVP SIHPGVTCDGCQMFPINGSRFKCRNCDDFDFCETCFKTKKHNTRH TFGRINEPGQSAVFCGRSGKQLKRCHSSQPGMLLDSWSRMVKSLN VSSSVNQASRLIDGSEPCWQSSGSQGKHWIRLEIFPDVLVHRLKM IVDPADSSYMPSLVVVSGGNSLNNLIELKTININPSDTTVPLLND CTEYHRYIEIAIKQCRSSGIDCKIHGLILLGRIRAEEEDLAAVPE LASDNEEEEDEKGNSGSLIRKKAAGLESAATIRTKVFVWGLNDKD QLGGLKGSKIKVPSFSETLSALNVVQVAGGSKSLFAVTVEGKVYA CGEATNGRLGLGISSGTVPIPRQITALSSYVVKKVAVHSGGRHAT ALTVDGKVFSWGEGDDGKLGHFSRMNCDKPRLIEALKTKRIRDIA CGSSHSAALTSSGELYTWGLGEYGRLGHGDNTTQLKPKMVKVLLG HRVIQVACGSRDAQTLALTDEGLVESWGDGDFGKLGRGGSEGCNI PQNIERLNGQGVCQIECGAQFSLALTKSGVVWTWGKGDYFRLGHG SDVHVRKPQVVEGLRGKKIVHVAVGALHCLAVTDSGQVYAWGDND HGQQGNGTTTVNRKPTLVQGLEGQKITRVACGSSHSVAWTTVDVA TPSVHEPVLFQTARDPLGASYLGVPSDADSSAASNKISGASNSKP NRPSLAKILLSLDGNLAKQQALSHILTALQIMYARDAVVGALMPA AMIAPVECPSFSSAAPSDASAMASPMNGEECMLAVDIEDRLSPNP WQEKREIVSSEDAVTPSAVTPSAPSASARPFIPVTDDLGAASIIA ETMTKTKEDVESQNKAAGPEPQALDEFTSLLIADDTRVVVDLLKL SVCSRAGDRGRDVLSAVLSGMGTAYPQVADMLLELCVTELEDVAT DSQSGRLSSQPVVVESSHPYTDDTSTSGTVKIPGAEGLRVEFDRQ CSTERRHDPLTVMDGVNRIVSVRSGREWSDWSSELRIPGDELKWK FISDGSVNGWGWRFTVYPIMPAAGPKELLSDRCVLSCPSMDLVTC LLDERLNLASNRSIVPRLAASLAACAQLSALAASHRMWALQRLRK LLTTEFGQSININRLLGENDGETRALSFTGSALAALVKGLPEALQ RQFEYEDPIVRGGKQLLHSPFFKVLVALACDLELDTLPCCAETHK WAWERRYCMASRVAVALDKRTPLPRLELDEVAKKIRELMADSENM DVLHESHDIFKREQDEQLVQWMNRRPDDWTLSAGGSGTIYGWGHN HRGQLGGIEGAKVKVPTPCEALATLRPVQLIGGEQTLFAVTADGK LYATGYGAGGRLGIGGTESVSTPTLLESIQHVFIKKVAVNSGGKH CLALSSEGEVYSWGEAEDGKLGHGNRSPCDRPRVIESLRGIEVVD VAAGGAHSACVTAAGDLYTWGKGRYGRLGHSDSEDQLKPKLVEAL QGHRVVDIACGSGDAQTLCLTDDDTVWSWGDGDYGKLGRGGSDGC KVPMKIDSLTGLGVVKVECGSQFSVALTKSGAVYTWGKGDYHRLG HGSDDHVRRPRQVQGLQGKKVIAIATGSLHCVCCTEDGEVYTWGD NDEGQLGDGTTNAIQRPRLVAALQGKKVNRVACGSAHTLAWSTSK PASAGKLPAQVPMEYNHLQEIPIIALRNRLLLLHHLSELFCPCIP MFDLEGSLDETGLGPSVGEDTLRGILISQGKEAAFRKVVQATMVR DRQHGPVVELNRIQVKRSRSKGGLAGPDGTKSVFGQMCAKMSSFG PDSLLLPHRVWKVKFVGESVDDCGGGYSESIAEICEELQNGLTPL LIVTPNGRDESGANRDCYLLSPAARAPVHSSMERFLGVLLGIAIR TGSPLSLNLAEPVWKQLAGMSLTIADLSEVDKDFIPGLMYIRDNE ATSEEFEAMSLPFTVPSASGQDIQLSSKHTHITLDNRAEYVRLAI NYRLHEFDEQVAAVREGMARVVPVPLLSLFTGYELETMVCGSPDI PLHLLKSVATYKGIEPSASLIQWFWEVMESESNTERSLFLRFVWG RTRLPRTIADFRGRDFVIQVLDKYNPPDHELPESYTCFFLLKLPR YSCKQVLEEKLKYAIHFCKSIDTDDYARIALTGEPAADDSSDDSD NEDVDSFASDSTQDYLTGH 99 BIN1 (SH3_9) MAEMGSKGVTAGKIASNVQKKLTRAQEKVLQKLGKADETKDEQFE QCVQNFNKQLTEGTRLQKDLRTYLASVKAMHEASKKLNECLQEVY EPDWPGRDEANKIAENNDLLWMDYHQKLVDQALLTMDTYLGQFPD IKSRIAKRGRKLVDYDSARHHYESLQTAKKKDEAKIAKPVSLLEK AAPQWCQGKLQAHLVAQTNLLRNQAEEELIKAQKVFEEMNVDLQE ELPSLWNSRVGFYVNTFQSIAGLEENFHKEMSKLNQNLNDVLVGL EKQHGSNTFTVKAQPSDNAPAKGNKSPSPPDGSPAATPEIRVNHE PEPAGGATPGATLPKSPSQLRKGPPVPPPPKHTPSKEVKQEQILS LFEDTFVPEISVTTPSQFEAPGPFSEQASLLDLDEDPLPPVTSPV KAPTPSGQSIPWDLWEPTESPAGSLPSGEPSAAEGTFAVSWPSQT AEPGPAQPAEASEVAGGTQPAAGAQEPGETAASEAASSSLPAVVV ETFPATVNGTVEGGSGAGRLDLPPGEMFKVQAQHDYTATDTDELQ LKAGDVVLVIPFQNPEEQDEGWLMGVKESDWNQHKELEKCRGVFP ENFTERVP 100 PCGF2 (RING MHRTTRIKITELNPHLMCALCGGYFIDATTIVECLHSFCKTCIVR finger protein YLETNKYCPMCDVQVHKTRPLLSIRSDKTLQDIVYKLVPGLFKDE domain) MKRRRDFYAAYPLTEVPNGSNEDRGEVLEQEKGALSDDEIVSLSI EFYEGARDRDEKKGPLENGDGDKEKTGVRFLRCPAAMTVMHLAKE LRNKMDVPSKYKVEVLYEDEPLKEYYTLMDIAYIYPWRRNGPLPL KYRVQPACKRLTLATVPTPSEGTNTSGASECESVSDKAPSPATLP ATSSSLPSPATPSHGSPSSHGPPATHPTSPTPPSTASGATTAANG GSLNCLQTPSSTSRGRKMTVNGAPVPPLT 101 TOX (HMG box) MDVRFYPPPAQPAAAPDAPCLGPSPCLDPYYCNKEDGENMYMSMT EPSQDYVPASQSYPGPSLESEDENIPPITPPSLPDHSLVHLNEVE SGYHSLCHPMNHNGLLPFHPQNMDLPEITVSNMLGQDGTLLSNSI SVMPDIRNPEGTQYSSHPQMAAMRPRGQPADIRQQPGMMPHGQLT TINQSQLSAQLGLNMGGSNVPHNSPSPPGSKSATPSPSSSVHEDE GDDTSKINGGEKRPASDMGKKPKTPKKKKKKDPNEPQKPVSAYAL FFRDTQAAIKGQNPNATFGEVSKIVASMWDGLGEEQKQVYKKKTE AAKKEYLKQLAAYRASLVSKSYSEPVDVKTSQPPQLINSKPSVFH GPSQAHSALYLSSHYHQQPGMNPHLTAMHPSLPRNIAPKPNNQMP VTVSIANMAVSPPPPLQISPPLHQHLNMQQHQPLTMQQPLGNQLP MQVQSALHSPTMQQGFTLQPDYQTIINPTSTAAQVVTQAMEYVRS GCRNPPPQPVDWNNDYCSSGGMQRDKALYLT 102 FOXA1 (HNF3A C- MLGTVKMEGHETSDWNSYYADTQEAYSSVPVSNMNSGLGSMNSMN terminal domain) TYMTMNTMTTSGNMTPASFNMSYANPGLGAGLSPGAVAGMPGGSA GAMNSMTAAGVTAMGTALSPSGMGAMGAQQAASMNGLGPYAAAMN PCMSPMAYAPSNLGRSRAGGGGDAKTFKRSYPHAKPPYSYISLIT MAIQQAPSKMLTLSEIYQWIMDLFPYYRQNQQRWQNSIRHSLSEN DCFVKVARSPDKPGKGSYWTLHPDSGNMFENGCYLRRQKRFKCEK QPGAGGGGGSGSGGSGAKGGPESRKDPSGASNPSADSPLHRGVHG KTGQLEGAPAPGPAASPQTLDHSGATATGGASELKTPASSTAPPI SSGPGALASVPASHPAHGLAPHESQLHLKGDPHYSENHPESINNL MSSSEQQHKLDEKAYEQALQYSPYGSTLPASLPLGSASVTTRSPI EPSALEPAYYQGVYSRPVLNTS 103 FOXA2 (HNF3B C- MLGAVKMEGHEPSDWSSYYAEPEGYSSVSNMNAGLGMNGMNTYMS terminal domain) MSAAAMGSGSGNMSAGSMNMSSYVGAGMSPSLAGMSPGAGAMAGM GGSAGAAGVAGMGPHLSPSLSPLGGQAAGAMGGLAPYANMNSMSP MYGQAGLSRARDPKTYRRSYTHAKPPYSYISLITMAIQQSPNKML TLSEIYQWIMDLFPFYRQNQQRWQNSIRHSLSENDCFLKVPRSPD KPGKGSFWTLHPDSGNMFENGCYLRRQKRFKCEKQLALKEAAGAA GSGKKAAAGAQASQAQLGEAAGPASETPAGTESPHSSASPCQEHK RGGLGELKGTPAAALSPPEPAPSPGQQQQAAAHLLGPPHHPGLPP EAHLKPEHHYAFNHPFSINNLMSSEQQHHHSHHHHQPHKMDLKAY EQVMHYPGYGSPMPGSLAMGPVTNKTGLDASPLAADTSYYQGVYS RPIMNSS 104 IRF2BP1 (IRF- MASVQASRRQWCYLCDLPKMPWAMVWDESEAVCRGCVNFEGADRI 2BP1 2 N-terminal ELLIDAARQLKRSHVLPEGRSPGPPALKHPATKDLAAAAAQGPQL domain) PPPQAQPQPSGTGGGVSGQDRYDRATSSGRLPLPSPALEYTLGSR LANGLGREEAVAEGARRALLGSMPGLMPPGLLAAAVSGLGSRGLT LAPGLSPARPLFGSDFEKEKQQRNADCLAELNEAMRGRAEEWHGR PKAVREQLLALSACAPFNVREKKDHGLVGRVFAFDATARPPGYEF ELKLFTEYPCGSGNVYAGVLAVARQMFHDALREPGKALASSGFKY LEYERRHGSGEWRQLGELLTDGVRSFREPAPAEALPQQYPEPAPA ALCGPPPRAPSRNLAPTPRRRKASPEPEGEAAGKMTTEEQQQRHW VAPGGPYSAETPGVPSPIAALKNVAEALGHSPKDPGGGGGPVRAG GASPAASSTAQPPTQHRLVARNGEAEVSPTAGAEAVSGGGSGTGA TPGAPLCCTLCRERLEDTHFVQCPSVPGHKFCFPCSREFIKAQGP AGEVYCPSGDKCPLVGSSVPWAFMQGEIATILAGDIKVKKERDP 105 IRF2BP2 (IRF- MAAAVAVAAASRRQSCYLCDLPRMPWAMIWDFTEPVCRGCVNYEG 2BP1 2 N-terminal ADRVEFVIETARQLKRAHGCFPEGRSPPGAAASAAAKPPPLSAKD domain) ILLQQQQQLGHGGPEAAPRAPQALERYPLAAAAERPPRLGSDEGS SRPAASLAQPPTPQPPPVNGILVPNGFSKLEEPPELNRQSPNPRR GHAVPPTLVPLMNGSATPLPTALGLGGRAAASLAAVSGTAAASLG SAQPTDLGAHKRPASVSSSAAVEHEQREAAAKEKQPPPPAHRGPA DSLSTAAGAAELSAEGAGKSRGSGEQDWVNRPKTVRDTLLALHQH GHSGPFESKEKKEPALTAGRLLGFEANGANGSKAVARTARKRKPS PEPEGEVGPPKINGEAQPWLSTSTEGLKIPMTPTSSFVSPPPPTA SPHSNRTTPPEAAQNGQSPMAALILVADNAGGSHASKDANQVHST TRRNSNSPPSPSSMNQRRLGPREVGGQGAGNTGGLEPVHPASLPD SSLATSAPLCCTLCHERLEDTHEVQCPSVPSHKFCFPCSRQSIKQ QGASGEVYCPSGEKCPLVGSNVPWAFMQGEIATILAGDVKVKKER DS 106 IRF2BPL IRF- MSAAQVSSSRRQSCYLCDLPRMPWAMIWDESEPVCRGCVNYEGAD 2BP1_2 N-terminal RIEFVIETARQLKRAHGCFQDGRSPGPPPPVGVKTVALSAKEAAA domain AAAAAAAAAAAAQQQQQQQQQQQQQQQQQQQQQQQQQLNHVDGSS KPAVLAAPSGLERYGLSAAAAAAAAAAAAVEQRSRFEYPPPPVSL GSSSHTARLPNGLGGPNGFPKPTPEEGPPELNRQSPNSSSAAASV ASRRGTHGGLVTGLPNPGGGGGPQLTVPPNLLPQTLLNGPASAAV LPPPPPHALGSRGPPTPAPPGAPGGPACLGGTPGVSATSSSASSS TSSSVAEVGVGAGGKRPGSVSSTDQERELKEKQRNAEALAELSES LRNRAEEWASKPKMVRDTLLTLAGCTPYEVRFKKDHSLLGRVFAF DAVSKPGMDYELKLFIEYPTGSGNVYSSASGVAKQMYQDCMKDFG RGLSSGFKYLEYEKKHGSGDWRLLGDLLPEAVRFFKEGVPGADML PQPYLDASCPMLPTALVSLSRAPSAPPGTGALPPAAPSGRGAAAS LRKRKASPEPPDSAEGALKLGEEQQRQQWMANQSEALKLTMSAGG FAAPGHAAGGPPPPPPPLGPHSNRTTPPESAPQNGPSPMAALMSV ADTLGTAHSPKDGSSVHSTTASARRNSSSPVSPASVPGQRRLASR NGDLNLQVAPPPPSAHPGMDQVHPQNIPDSPMANSGPLCCTICHE RLEDTHEVQCPSVPSHKFCFPCSRESIKAQGATGEVYCPSGEKCP LVGSNVPWAFMQGEIATILAGDVKVKKERDP 107 HOXA13 MTASVLLHPRWIEPTVMELYDNGGGLVADELNKNMEGAAAAAAAA (homeodomain) AAAAAAGAGGGGFPHPAAAAAGGNESVAAAAAAAAAAAANQCRNL MAHPAPLAPGAASAYSSAPGEAPPSAAAAAAAAAAAAAAAAAASS SGGPGPAGPAGAEAAKQCSPCSAAAQSSSGPAALPYGYFGSGYYP CARMGPHPNAIKSCAQPASAAAAAAFADKYMDTAGPAAEEFSSRA KEFAFYHQGYAAGPYHHHQPMPGYLDMPVVPGLGGPGESRHEPLG LPMESYQPWALPNGWNGQMYCPKEQAQPPHLWKSTLPDVVSHPSD ASSYRRGRKKRVPYTKVQLKELEREYATNKFITKDKRRRISATTN LSERQVTIWFQNRRVKEKKVINKLKTTS 108 HOXB13 MEPGNYATLDGAKDIEGLLGAGGGRNLVAHSPLTSHPAAPTLMPA (homeodomain) VNYAPLDLPGSAEPPKQCHPCPGVPQGTSPAPVPYGYFGGGYYSC RVSRSSLKPCAQAATLAAYPAETPTAGEEYPSRPTEFAFYPGYPG TYQPMASYLDVSVVQTLGAPGEPRHDSLLPVDSYQSWALAGGWNS QMCCQGEQNPPGPFWKAAFADSSGQHPPDACAFRRGRKKRIPYSK GQLRELEREYAANKFITKDKRRKISAATSLSERQITIWFQNRRVK EKKVLAKVKNSATP 109 HOXC13 MTTSLLLHPRWPESLMYVYEDSAAESGIGGGGGGGGGTGGGAGGG (homeodomain) CSGASPGKAPSMDGLGSSCPASHCRDLLPHPVLGRPPAPLGAPQG AVYTDIPAPEAARQCAPPPAPPTSSSATLGYGYPFGGSYYGCRLS HNVNLQQKPCAYHPGDKYPEPSGALPGDDLSSRAKEFAFYPSFAS SYQAMPGYLDVSVVPGISGHPEPRHDALIPVEGYQHWALSNGWDS QVYCSKEQSQSAHLWKSPFPDVVPLQPEVSSYRRGRKKRVPYTKV QLKELEKEYAASKFITKEKRRRISATTNLSERQVTIWFQNRRVKE KKVVSKSKAPHLHST 110 HOXA11 MDFDERGPCSSNMYLPSCTYYVSGPDFSSLPSELPQTPSSRPMTY (homeodomain) SYSSNLPQVQPVREVTFREYAIEPATKWHPRGNLAHCYSAEELVH RDCLQAPSAAGVPGDVLAKSSANVYHHPTPAVSSNFYSTVGRNGV LPQAFDQFFETAYGTPENLASSDYPGDKSAEKGPPAATATSAAAA AAATGAPATSSSDSGGGGGCRETAAAAEEKERRRRPESSSSPESS SGHTEDKAGGSSGQRTRKKRCPYTKYQIRELEREFFFSVYINKEK RLQLSRMLNLTDRQVKIWFQNRRMKEKKINRDRLQYYSANPLL 111 HOXC11 MENSVNLGNFCSPSRKERGADEGERGSCASNLYLPSCTYYMPEFS (homeodomain) TVSSFLPQAPSRQISYPYSAQVPPVREVSYGLEPSGKWHHRNSYS SCYAAADELMHRECLPPSTVTEILMKNEGSYGGHHHPSAPHATPA GFYSSVNKNSVLPQAFDRFFDNAYCGGGDPPAEPPCSGKGEAKGE PEAPPASGLASRAEAGAEAEAEEENTNPSSSGSAHSVAKEPAKGA APNAPRTRKKRCPYSKFQIRELEREFFENVYINKEKRLQLSRMLN LTDRQVKIWFQNRRMKEKKLSRDRLQYFSGNPLL 112 HOXC10 MTCPRNVTPNSYAEPLAAPGGGERYSRSAGMYMQSGSDENCGVMR (homeodomain) GCGLAPSLSKRDEGSSPSLALNTYPSYLSQLDSWGDPKAAYRLEQ PVGRPLSSCSYPPSVKEENVCCMYSAEKRAKSGPEAALYSHPLPE SCLGEHEVPVPSYYRASPSYSALDKTPHCSGANDFEAPFEQRASL NPRAEHLESPQLGGKVSFPETPKSDSQTPSPNEIKTEQSLAGPKG SPSESEKERAKAADSSPDTSDNEAKEEIKAENTTGNWLTAKSGRK KRCPYTKHQTLELEKEFLENMYLTRERRLEISKTINLTDRQVKIW FQNRRMKLKKMNRENRIRELTSNENFT 113 HOXA10 MSARKGYLLPSPNYPTTMSCSESPAANSFLVDSLISSGRGEAGGG (homeodomain) GGGAGGGGGGGYYAHGGVYLPPAADLPYGLQSCGLFPTLGGKRNE AASPGSGGGGGGLGPGAHGYGPSPIDLWLDAPRSCRMEPPDGPPP PPQQQPPPPPQPPQPAPQATSCSFAQNIKEESSYCLYDSADKCPK VSATAAELAPFPRGPPPDGCALGTSSGVPVPGYFRLSQAYGTAKG YGSGGGGAQQLGAGPFPAQPPGRGFDLPPALASGSADAARKERAL DSPPPPTLACGSGGGSQGDEEAHASSSAAEELSPAPSESSKASPE KDSLGNSKGENAANWLTAKSGRKKRCPYTKHQTLELEKEFLENMY LTRERRLEISRSVHLTDRQVKIWFQNRRMKLKKMNRENRIRELTA NENES 114 HOXB9 MSISGTLSSYYVDSIISHESEDAPPAKFPSGQYASSRQPGHAEHL (homeodomain) EFPSCSFQPKAPVFGASWAPLSPHASGSLPSVYHPYIQPQGVPPA ESRYLRTWLEPAPRGEAAPGQGQAAVKAEPLLGAPGELLKQGTPE YSLETSAGREAVLSNQRPGYGDNKICEGSEDKERPDQTNPSANWL HARSSRKKRCPYTKYQTLELEKEFLENMYLTRDRRHEVARLLNLS ERQVKIWFQNRRMKMKKMNKEQGKE 115 HOXA9 MATTGALGNYYVDSFLLGADAADELSVGRYAPGTLGQPPRQAATL (homeodomain) AEHPDFSPCSFQSKATVEGASWNPVHAAGANAVPAAVYHHHHHHP YVHPQAPVAAAAPDGRYMRSWLEPTPGALSFAGLPSSRPYGIKPE PLSARRGDCPTLDTHTLSLTDYACGSPPVDREKQPSEGAFSENNA ENESGGDKPPIDPNNPAANWLHARSTRKKRCPYTKHQTLELEKEF LFNMYLTRDRRYEVARLLNLTERQVKIWFQNRRMKMKKINKDRAK DE 116 ZFP28_HUMAN NKKLEAVGTGIEPKAMSQGLVTFGDVAVDESQEEWEWLNPIQRNL YRKVMLENYRNLASLGLCVSKPDVISSLEQGKEPW 117 ZN334_HUMAN KMKKFQIPVSFQDLTVNFTQEEWQQLDPAQRLLYRDVMLENYSNL VSVGYHVSKPDVIFKLEQGEEPWIVEEFSNQNYPD 118 ZN568_HUMAN CSQESALSEEEEDTTRPLETVTFKDVAVDLTQEEWEQMKPAQRNL YRDVMLENYSNLVTVGCQVTKPDVIFKLEQEEEPW 119 ZN37A_HUMAN ITSQGSVSFRDVTVGFTQEEWQHLDPAQRTLYRDVMLENYSHLVS VGYCIPKPEVILKLEKGEEPWILEEKFPSQSHLEL 120 ZN181_HUMAN PQVTFNDVAIDFTHEEWGWLSSAQRDLYKDVMVQNYENLVSVAGL SVTKPYVITLLEDGKEPWMMEKKLSKGMIPDWESR 121 ZN510_HUMAN PLRFSTLFQEQQKMNISQASVSFKDVTIEFTQEEWQQMAPVQKNL YRDVMLENYSNLVSVGYCCFKPEVIFKLEQGEEPW 122 ZN862_HUMAN QDPSAEGLSEEVPVVFEELPVVFEDVAVYFTREEWGMLDKRQKEL YRDVMRMNYELLASLGPAAAKPDLISKLERRAAPW 123 ZN140_HUMAN SQGSVTFRDVAIDFSQEEWKWLQPAQRDLYRCVMLENYGHLVSLG LSISKPDVVSLLEQGKEPWLGKREVKRDLFSVSES 124 ZN208_HUMAN GSLTFRDVAIEFSLEEWQCLDTAQQNLYRNVMLENYRNLVELGIA AFKPDLIIFLEEGKESWNMKRHEMVEESPVICSHE 125 ZN248_HUMAN NKSQEQVSFKDVCVDFTQEEWYLLDPAQKILYRDVILENYSNLVS VGYCITKPEVIFKIEQGEEPWILEKGFPSQCHPER 126 ZN571_HUMAN PHLLVTERDVAIDESQEEWECLDPAQRDLYRDVMLENYSNLISLD LESSCVTKKLSPEKEIYEMESLQWENMGKRINHHL 127 ZN699_HUMAN EEERKTAELQKNRIQDSVVFEDVAVDETQEEWALLDLAQRNLYRD VMLENFQNLASLGYPLHTPHLISQWEQEEDLQTVK 128 ZN726_HUMAN GLLTFRDVAIEFSLEEWQCLDTAQKNLYRNVMLENYRNLAFLGIA VSKPDLIICLEKEKEPWNMKRDEMVDEPPGICPHE 129 ZIKI_HUMAN RAPTQVTVSPETHMDLTKGCVTFEDIAIYFSQDEWGLLDEAQRLL YLEVMLENFALVASLGCGHGTEDEETPSDQNVSVG 130 ZNF2_HUMAN AAVSPTTRCQESVTFEDVAVVETDEEWSRLVPIQRDLYKEVMLEN YNSIVSLGLPVPQPDVIFQLKRGDKPWMVDLHGSE 131 Z705F_HUMAN HSLEKVTFEDVAIDFTQEEWDMMDTSKRKLYRDVMLENISHLVSL GYQISKSYIILQLEQGKELWREGRVFLQDQNPDRE 132 ZNF14_HUMAN DSVSFEDVAVNFTLEEWALLDSSQKKLYEDVMQETEKNLVCLGKK WEDQDIEDDHRNQGKNRRCHMVERLCESRRGSKCG 133 ZN471_HUMAN NVEVVKVMPQDLVTFKDVAIDESQEEWQWMNPAQKRLYRSMMLEN YQSLVSLGLCISKPYVISLLEQGREPWEMTSEMTR 134 ZN624_HUMAN TQPDEDLHLQAEETQLVKESVTFKDVAIDFTLEEWRLMDPTQRNL HKDVMLENYRNLVSLGLAVSKPDMISHLENGKGPW 135 ZNF84_HUMAN TMLQESFSFDDLSVDFTQKEWQLLDPSQKNLYKDVMLENYSSLVS LGYEVMKPDVIFKLEQGEEPWVGDGEIPSSDSPEV 136 ZNF7_HUMAN EVVTFGDVAVHFSREEWQCLDPGQRALYREVMLENHSSVAGLAGE LVFKPELISRLEQGEEPWVLDLQGAEGTEAPRTSK 137 ZN891_HUMAN RNAEEERMIAVELTTWLQEPMTFKDVAVEFTQEEWMMLDSAQRSL YRDVMLENYRNLTSVEYQLYRLTVISPLDQEEIRN 138 ZN337_HUMAN GPQGARRQAFLAFGDVTVDFTQKEWRLLSPAQRALYREVTLENYS HLVSLGILHSKPELIRRLEQGEVPWGEERRRRPGP 139 Z705G_HUMAN HSLKKLTFEDVAIDFTQEEWAMMDTSKRKLYRDVMLENISHLVSL GYQISKSYIILQLEQGKELWREGRVFLQDQNPNRE 140 ZN529_HUMAN MPEVEFPDQFFTVLTMDHELVTLRDVVINESQEEWEYLDSAQRNL YWDVMMENYSNLLSLDLESRNETKHLSVGKDIIQN 141 ZN729_HUMAN PGAPGSLEMGPLTFRDVTIEFSLEEWQCLDTVQQNLYRDVMLENY RNLVFLGMAVFKPDLITCLKQGKEPWNMKRHEMVT 142 ZN419_HUMAN RDPAQVPVAADLLTDHEEGYVTFEDVAVYFSQEEWRLLDDAQRLL YRNVMLENFTLLASLGLASSKTHEITQLESWEEPF 143 Z705A_HUMAN HSLKKVTFEDVAIDFTQEEWAMMDTSKRKLYRDVMLENISHLVSL GYQISKSYIILQLEQGKELWREGREFLQDQNPDRE 144 ZNF45_HUMAN TKSKEAVTFKDVAVVESEEELQLLDLAQRKLYRDVMLENERNVVS VGHQSTPDGLPQLEREEKLWMMKMATQRDNSSGAK 145 ZN302_HUMAN SQVTFSDVAIDESHEEWACLDSAQRDLYKDVMVQNYENLVSVGLS VTKPYVIMLLEDGKEPWMMEKKLSKAYPFPLSHSV 146 ZN486_HUMAN PGPLRSLEMESLQFRDVAVEFSLEEWHCLDTAQQNLYRDVMLENY RHLVELGIIVSKPDLITCLEQGIKPLTMKRHEMIA 147 ZN621_HUMAN LQTTWPQESVTFEDVAVYFTQNQWASLDPAQRALYGEVMLENYAN VASLVAFPFPKPALISHLERGEAPWGPDPWDTEIL 148 ZN688_HUMAN APLLAPRPGETRPGCRKPGTVSFADVAVYESPEEWGCLRPAQRAL YRDVMQETYGHLGALGFPGPKPALISWMEQESEAW 149 ZN33A_HUMAN NKVEQKSQESVSFKDVTVGFTQEEWQHLDPSQRALYRDVMLENYS NLVSVGYCVHKPEVIFRLQQGEEPWKQEEEFPSQS 150 ZN554_HUMAN CFSQEERMAAGYLPRWSQELVTFEDVSMDESQEEWELLEPAQKNL YREVMLENYRNVVSLEALKNQCTDVGIKEGPLSPA 151 ZN878_HUMAN DSVAFEDVAVNFTQEEWALLDPSQKNLYREVMQETLRNLTSIGKK WNNQYIEDEHQNPRRNLRRLIGERLSESKESHQHG 152 ZN772_HUMAN MGPAQVPMNSEVIVDPIQGQVNFEDVEVYFSQEEWVLLDEAQRLL YRDVMLENFALMASLGHTSFMSHIVASLVMGSEPW 153 ZN224_HUMAN TTFKEAMTFKDVAVVFTEEELGLLDLAQRKLYRDVMLENERNLLS VGHQAFHRDTFHFLREEKIWMMKTAIQREGNSGDK 154 ZN184_HUMAN DSTLLQGGHNLLSSASFQEAVTFKDVIVDFTQEEWKQLDPGQRDL FRDVTLENYTHLVSIGLQVSKPDVISQLEQGTEPW 155 ZN544_HUMAN EARSMLVPPQASVCFEDVAMAFTQEEWEQLDLAQRTLYREVTLET WEHIVSLGLFLSKSDVISQLEQEEDLCRAEQEAPR 156 ZNF57_HUMAN DSVVFEDVAVDETLEEWALLDSAQRDLYRDVMLETERNLASVDDG TQFKANGSVSLQDMYGQEKSKEQTIPNETGNNSCA 157 ZN283_HUMAN EESHGALISSCNSRTMTDGLVTERDVAIDESQEEWECLDPAQRDL YVDVMLENYSNLVSLDLESKTYETKKIFSENDIFE 158 ZN549_HUMAN VITPQIPMVTEEFVKPSQGHVTFEDIAVYFSQEEWGLLDEAQRCL YHDVMLENFSLMASVGCLHGIEAEEAPSEQTLSAQ 159 ZN211_HUMAN VQLRPQTRMATALRDPASGSVTFEDVAVYESWEEWDLLDEAQKHL YFDVMLENFALTSSLGCWCGVEHEETPSEQRISGE 160 ZN615_HUMAN MQAQESLTLEDVAVDFTWEEWQFLSPAQKDLYRDVMLENYSNLVA VGYQASKPDALSKLERGEETCTTEDEIYSRICSEI 161 ZN253_HUMAN GPLQFRDVAIEFSLEEWHCLDTAQRNLYRDVMLENYRNLVFLGIV VSKPDLVTCLEQGKKPLTMERHEMIAKPPVMSSHF 162 ZN226_HUMAN NMFKEAVTFKDVAVAFTEEELGLLGPAQRKLYRDVMVENERNLLS VGHPPFKQDVSPIERNEQLWIMTTATRRQGNLGEK 163 ZN730_HUMAN GALTFRDVAIEFSLEEWQCLDTEQQNLYRNVMLDNYRNLVELGIA VSKPDLITCLEQEKEPWNLKTHDMVAKPPVICSHI 164 Z585A_HUMAN SPQKSSALAPEDHGSSYEGSVSERDVAIDESREEWRHLDPSQRNL YRDVMLETYSHLLSVGYQVPEAEVVMLEQGKEPWA 165 ZN732_HUMAN ELLTFRDVAIEFSPEEWKCLDPAQQNLYRDVMLENYRNLISLGVA ISNPDLVIYLEQRKEPYKVKIHETVAKHPAVCSHF 166 ZN681_HUMAN EPLKERDVAIEFSLEEWQCLDTIQQNLYRNVMLENYRNLVELGIV VSKPDLITCLEQEKEPWTRKRHRMVAEPPVICSHE 167 ZN667_HUMAN PSARGKSKSKAPITFGDLAIYFSQEEWEWLSPIQKDLYEDVMLEN YRNLVSLGLSFRRPNVITLLEKGKAPWMVEPVRRR 168 ZN649_HUMAN TKAQESLTLEDVAVDFTWEEWQFLSPAQKDLYRDVMLENYSNLVS VGYQAGKPDALTKLEQGEPLWTLEDEIHSPAHPEI 169 ZN470_HUMAN SQEEVEVAGIKLCKAMSLGSVTFTDVAIDESQDEWEWLNLAQRSL YKKVMLENYRNLVSVGLCISKPDVISLLEQEKDPW 170 ZN484_HUMAN TKSLESVSFKDVTVDESRDEWQQLDLAQKSLYREVMLENYENLIS VGCQVPKPEVIFSLEQEEPCMLDGEIPSQSRPDGD 171 ZN431_HUMAN SGCPGAERNLLVYSYFEKETLTERDVAIEFSLEEWECLNPAQQNL YMNVMLENYKNLVELGVAVSKQDPVTCLEQEKEPW 172 ZN382_HUMAN PLQGSVSFKDVTVDFTQEEWQQLDPAQKALYRDVMLENYCHFVSV GFHMAKPDMIRKLEQGEELWTQRIFPSYSYLEEDG 173 ZN254_HUMAN PGPPRSLEMGLLTERDVAIEFSLEEWQHLDIAQQNLYRNVMLENY RNLAFLGIAVSKPDLITCLEQGKEPWNMKRHEMVD 174 ZN124_HUMAN SGHPGSWEMNSVAFEDVAVNFTQEEWALLDPSQKNLYRDVMQETF RNLASIGNKGEDQSIEDQYKNSSRNLRHIISHSGN 175 ZN607_HUMAN SYGSITFGDVAIDFSHQEWEYLSLVQKTLYQEVMMENYDNLVSLA GHSVSKPDLITLLEQGKEPWMIVREETRGECTDLD 176 ZN317_HUMAN DLFVCSGLEPHTPSVGSQESVTFQDVAVDFTEKEWPLLDSSQRKL YKDVMLENYSNLTSLGYQVGKPSLISHLEQEEEPR 177 ZN620_HUMAN FQTAWRQEPVTFEDVAVYFTQNEWASLDSVQRALYREVMLENYAN VASLAFPFTTPVLVSQLEQGELPWGLDPWEPMGRE 178 ZN141_HUMAN ELLTFRDVAIEFSPEEWKCLDPDQQNLYRDVMLENYRNLVSLGVA ISNPDLVTCLEQRKEPYNVKIHKIVARPPAMCSHE 179 ZN584_HUMAN AGEAEAQLDPSLQGLVMFEDVTVYFSREEWGLLNVTQKGLYRDVM LENFALVSSLGLAPSRSPVFTQLEDDEQSWVPSWV 180 ZN540_HUMAN AHALVTERDVAIDFSQKEWECLDTTQRKLYRDVMLENYNNLVSLG YSGSKPDVITLLEQGKEPCVVARDVTGRQCPGLLS 181 ZN75D_HUMAN KRIKHWKMASKLILPESLSLLTFEDVAVYFSEEEWQLLNPLEKTL YNDVMQDIYETVISLGLKLKNDTGNDHPISVSTSE 182 ZN555_HUMAN DSVVFEDVAVDFTLEEWALLDSAQRDLYRDVMLETFQNLASVDDE TQFKASGSVSQQDIYGEKIPKESKIATFTRNVSWA 183 ZN658_HUMAN NMSQASVSFQDVTVEFTREEWQHLGPVERTLYRDVMLENYSHLIS VGYCITKPKVISKLEKGEEPWSLEDEFLNQRYPGY 184 ZN684_HUMAN ISFQESVTFQDVAVDFTAEEWQLLDCAERTLYWDVMLENYRNLIS VGCPITKTKVILKVEQGQEPWMVEGANPHESSPES 185 RBAK_HUMAN NTLQGPVSFKDVAVDFTQEEWQQLDPDEKITYRDVMLENYSHLVS VGYDTTKPNVIIKLEQGEEPWIMGGEFPCQHSPEA 186 ZN829_HUMAN HPEEEERMHDELLQAVSKGPVMFRDVSIDESQEEWECLDADQMNL YKEVMLENFSNLVSVGLSNSKPAVISLLEQGKEPW 187 ZN582_HUMAN SLGSELFRDVAIVFSQEEWQWLAPAQRDLYRDVMLETYSNLVSLG LAVSKPDVISFLEQGKEPWMVERVVSGGLCPVLES 188 ZN112_HUMAN TKFQEMVTFKDVAVVFTEEELGLLDSVQRKLYRDVMLENERNLLL VAHQPFKPDLISQLEREEKLLMVETETPRDGCSGR 189 ZN716_HUMAN AKRPGPPGSREMGLLTFRDIAIEFSLAEWQCLDHAQQNLYRDVML ENYRNLVSLGIAVSKPDLITCLEQNKEPQNIKRNE 190 HKR1_HUMAN TCMVHRQTMSCSGAGGITAFVAFRDVAVYFTQEEWRLLSPAQRTL HREVMLETYNHLVSLEIPSSKPKLIAQLERGEAPW 191 ZN350_HUMAN IQAQESITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLVA VGYQASKPDALFKLEQGEQLWTIEDGIHSGACSDI 192 ZN480_HUMAN AQKRRKRKAKESGMALPQGHLTERDVAIEFSQAEWKCLDPAQRAL YKDVMLENYRNLVSLGISLPDLNINSMLEQRREPW 193 ZN416_HUMAN DSTSVPVTAEAKLMGFTQGCVTFEDVAIYFSQEEWGLLDEAQRLL YRDVMLENFALITALVCWHGMEDEETPEQSVSVEG 194 ZNF92_HUMAN GPLTFRDVKIEFSLEEWQCLDTAQRNLYRDVMLENYRNLVELGIA VSKPDLITWLEQGKEPWNLKRHEMVDKTPVMCSHF 195 ZN100_HUMAN SGCPGAERSLLVQSYFEKGPLTERDVAIEFSLEEWQCLDSAQQGL YRKVMLENYRNLVFLAGIALTKPDLITCLEQGKEP 196 ZN736_HUMAN GVLTFRDVAVEFSPEEWECLDSAQQRLYRDVMLENYGNLVSLGLA IFKPDLMTCLEQRKEPWKVKRQEAVAKHPAGSFHF 197 ZNF74_HUMAN KENLEDISGWGLPEARSKESVSFKDVAVDETQEEWGQLDSPQRAL YRDVMLENYQNLLALGPPLHKPDVISHLERGEEPW 198 CBX1_HUMAN EESEKPRGFARGLEPERIIGATDSSGELMELMKWKNSDEADLVPA KEANVKCPQVVISFYEERLTWHSYPSEDDDKKDDK 199 ZN443_HUMAN ASVALEDVAVNFTREEWALLGPCQKNLYKDVMQETIRNLDCVVMK WKDQNIEDQYRYPRKNLRCRMLERFVESKDGTQCG 200 ZN195_HUMAN TLLTFRDVAIEFSLEEWKCLDLAQQNLYRDVMLENYRNLESVGLT VCKPGLITCLEQRKEPWNVKRQEAADGHPEMGFHH 201 ZN530_HUMAN AAALRAPTQQVEVAFEDVAIYFSQEEWELLDEMQRLLYRDVMLEN FAVMASLGCWCGAVDEGTPSAESVSVEELSQGRTP 202 ZN782_HUMAN NTFQASVSFQDVTVEFSQEEWQHMGPVERTLYRDVMLENYSHLVS VGYCFTKPELIFTLEQGEDPWLLEKEKGELSRNSP 203 ZN791_HUMAN DSVAFEDVSVSFSQEEWALLAPSQKKLYRDVMQETFKNLASIGEK WEDPNVEDQHKNQGRNLRSHTGERLCEGKEGSQCA 204 ZN331_HUMAN AQGLVTFADVAIDESQEEWACLNSAQRDLYWDVMLENYSNLVSLD LESAYENKSLPTEKNIHEIRASKRNSDRRSKSLGR 205 Z354C_HUMAN AVDLLSAQEPVTERDVAVFFSQDEWLHLDSAQRALYREVMLENYS SLVSLGIPFSMPKLIHQLQQGEDPCMVEREVPSDT 206 ZN157_HUMAN SPQRFPALIPGEPGRSFEGSVSFEDVAVDFTRQEWHRLDPAQRTM HKDVMLETYSNLASVGLCVAKPEMIFKLERGEELW 207 ZN727_HUMAN RVLTFRDVAVEFSPEEWECLDSAQQRLYRDVMLENYGNLESLGLA IFKPDLITYLEQRKEPWNARRQKTVAKHPAGSLHF 208 ZN550_HUMAN AETKDAAQMLVTFKDVAVTFTREEWRQLDLAQRTLYREVMLETCG LLVSLGHRVPKPELVHLLEHGQELWIVKRGLSHAT 209 ZN793_HUMAN IEYQIPVSFKDVVVGFTQEEWHRLSPAQRALYRDVMLETYSNLVS VGYEGTKPDVILRLEQEEAPWIGEAACPGCHCWED 210 ZN235_HUMAN TKFQEAVTFKDVAVAFTEEELGLLDSAQRKLYRDVMLENERNLVS VGHQSFKPDMISQLEREEKLWMKELQTQRGKHSGD 211 ZNF8_HUMAN DEGVAGVMSVGPPAARLQEPVTERDVAVDFTQEEWGQLDPTQRIL YRDVMLETFGHLLSIGPELPKPEVISQLEQGTELW 212 ZN724_HUMAN GPLTEMDVAIEFSVEEWQCLDTAQQNLYRNVMLENYRNLVELGIA VSKPDLITCLEQGKEPWNMERHEMVAKPPGMCCYF 213 ZN573_HUMAN HQVGLIRSYNSKTMTCFQELVTERDVAIDFSRQEWEYLDPNQRDL YRDVMLENYRNLVSLGGHSISKPVVVDLLERGKEP 214 ZN577_HUMAN NATIVMSVRREQGSSSGEGSLSFEDVAVGFTREEWQFLDQSQKVL YKEVMLENYINLVSIGYRGTKPDSLFKLEQGEPPG 215 ZN789_HUMAN FPPARGKELLSFEDVAMYFTREEWGHLNWGQKDLYRDVMLENYRN MVLLGFQFPKPEMICQLENWDEQWILDLPRTGNRK 216 ZN718_HUMAN ELLTFKDVAIEFSPEEWKCLDTSQQNLYRDVMLENYRNLVSLGVS ISNPDLVTSLEQRKEPYNLKIHETAARPPAVCSHE 217 ZN300_HUMAN MKSQGLVSFKDVAVDFTQEEWQQLDPSQRTLYRDVMLENYSHLVS MGYPVSKPDVISKLEQGEEPWIIKGDISNWIYPDE 218 ZN383_HUMAN AEGSVMFSDVSIDFSQEEWDCLDPVQRDLYRDVMLENYGNLVSMG LYTPKPQVISLLEQGKEPWMVGRELTRGLCSDLES 219 ZN429_HUMAN GPLTFTDVAIEFSLEEWQCLDTAQQNLYRNVMLENYRNLVELGIA VSKPDLITCLEKEKEPCKMKRHEMVDEPPVVCSHF 220 ZN677_HUMAN ALSQGLFTFKDVAIEFSQEEWECLDPAQRALYRDVMLENYRNLLS LDEDNIPPEDDISVGFTSKGLSPKENNKEELYHLV 221 ZN850_HUMAN NMEGLVMFQDLSIDESQEEWECLDAAQKDLYRDVMMENYSSLVSL GLSIPKPDVISLLEQGKEPWMVSRDVLGGWCRDSE 222 ZN454_HUMAN AVSHLPTMVQESVTFKDVAILFTQEEWGQLSPAQRALYRDVMLEN YSNLVSLGLLGPKPDTFSQLEKREVWMPEDTPGGF 223 ZN257_HUMAN GPLTIRDVTVEFSLEEWHCLDTAQQNLYRDVMLENYRNLVELGIA VSKPDLITCLEQGKEPCNMKRHEMVAKPPVMCSHI 224 ZN264_HUMAN AAAVLTDRAQVSVTEDDVAVTFTKEEWGQLDLAQRTLYQEVMLEN CGLLVSLGCPVPKAELICHLEHGQEPWTRKEDLSQ 225 ZFP82_HUMAN ALRSVMESDVSIDESPEEWEYLDLEQKDLYRDVMLENYSNLVSLG CFISKPDVISSLEQGKEPWKVVRKGRRQYPDLETK 226 ZFP14_HUMAN AHGSVTFRDVAIDFSQEEWEFLDPAQRDLYRDVMWENYSNFISLG PSISKPDVITLLDEERKEPGMVVREGTRRYCPDLE 227 ZN485_HUMAN APRAQIQGPLTFGDVAVAFTRIEWRHLDAAQRALYRDVMLENYGN LVSVGLLSSKPKLITQLEQGAEPWTEVREAPSGTH 228 ZN737_HUMAN GPLQFRDVAIEFSLEEWHCLDTAQRNLYRNVMLENYRNLVELGIV VSKPDLITCLEQGKKPLTMKKHEMVANPSVTCSHF 229 ZNF44_HUMAN TLPRGQPEVLEWGLPKDQDSVAFEDVAVNFTHEEWALLGPSQKNL YRDVMRETIRNLNCIGMKWENQNIDDQHQNLRRNP 230 ZN596_HUMAN PSPDSMTFEDIIVDFTQEEWALLDTSQRKLFQDVMLENISHLVSI GKQLCKSVVLSQLEQVEKLSTQRISLLQGREVGIK 231 ZN565_HUMAN EESREIRAGQIVLKAMAQGLVTERDVAIEFSLEEWKCLEPAQRDL YREVTLENFGHLASLGLSISKPDVVSLLEQGKEPW 232 ZN543_HUMAN AASAQVSVTFEDVAVTFTQEEWGQLDAAQRTLYQEVMLETCGLLM SLGCPLFKPELIYQLDHRQELWMATKDLSQSSYPG 233 ZFP69_HUMAN RESLEDEVTPGLPTAESQELLTFKDISIDFTQEEWGQLAPAHQNL YREVMLENYSNLVSVGYQLSKPSVISQLEKGEEPW 234 SUMO1_HUMAN EGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSL RFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGG 235 ZNF12_HUMAN NKSLGPVSFKDVAVDFTQEEWQQLDPEQKITYRDVMLENYSNLVS VGYHIIKPDVISKLEQGEEPWIVEGEFLLQSYPDE 236 ZN169_HUMAN SPGLLTTRKEALMAFRDVAVAFTQKEWKLLSSAQRTLYREVMLEN YSHLVSLGIAFSKPKLIEQLEQGDEPWREENEHLL 237 ZN433_HUMAN MFQDSVAFEDVAVTFTQEEWALLDPSQKNLCRDVMQETERNLASI GKKWKPQNIYVEYENLRRNLRIVGERLFESKEGHQ 238 SUMO3_HUMAN ENDHINLKVAGQDGSVVQFKIKRHTPLSKLMKAYCERQGLSMRQI RFREDGQPINETDTPAQLEMEDEDTIDVEQQQTGG 239 ZNF98_HUMAN PGPLGSLEMGVLTFRDVALEFSLEEWQCLDTAQQNLYRNVMLENY RNLVFVGIAASKPDLITCLEQGKEPWNVKRHEMVT 240 ZN175_HUMAN LSQKPQVLGPEKQDGSCEASVSFEDVTVDESREEWQQLDPAQRCL YRDVMLELYSHLFAVGYHIPNPEVIFRMLKEKEPR 241 ZN347_HUMAN ALTQGQVTFRDVAIEFSQEEWTCLDPAQRTLYRDVMLENYRNLAS LGISCEDLSIISMLEQGKEPFTLESQVQIAGNPDG 242 ZNF25_HUMAN NKFQGPVTLKDVIVEFTKEEWKLLTPAQRTLYKDVMLENYSHLVS VGYHVNKPNAVFKLKQGKEPWILEVEFPHRGFPED 243 ZN519_HUMAN ELLTFRDVAIEFSPEEWKCLDPAQQNLYRDVMLENYRNLVSLAVY SYYNQGILPEQGIQDSFKKATLGRYGSCGLENICL 244 Z585B_HUMAN SPQKSSALAPEDHGSSYEGSVSERDVAIDESREEWRHLDLSQRNL YRDVMLETYSHLLSVGYQVPKPEVVMLEQGKEPWA 245 ZIM3_HUMAN NNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLVS VGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAE 246 ZN517_HUMAN AMALPMPGPQEAVVFEDVAVYFTRIEWSCLAPDQQALYRDVMLEN YGNLASLGELVAKPALISLLEQGEEPGALILQVAE 247 ZN846_HUMAN DSSQHLVTFEDVAVDFTQEEWTLLDQAQRDLYRDVMLENYKNLII LAGSELFKRSLMSGLEQMEELRTGVTGVLQELDLQ 248 ZN230_HUMAN TTFKEAVTFKDVAVFFTEEELGLLDPAQRKLYQDVMLENFTNLLS VGHQPFHPFHFLREEKFWMMETATQREGNSGGKTI 249 ZNF66_HUMAN GPLQFRDVAIEFSLEEWHCLDMAQRNLYRDVMLENYRNLVELGIV VSKPDLITHLEQGKKPSTMQRHEMVANPSVLCSHF 250 ZFP1_HUMAN NKSQGSVSFTDVTVDFTQEEWEQLDPSQRILYMDVMLENYSNLLS VEVWKADDQMERDHRNPDEQARQFLILKNQTPIEE 251 ZN713_HUMAN EEEEMNDGSQMVRSQESLTFQDVAVDFTREEWDQLYPAQKNLYRD VMLENYRNLVALGYQLCKPEVIAQLELEEEWVIER 252 ZN816_HUMAN EEATKKSKEKEPGMALPQGRLTERDVAIEFSLEEWKCLNPAQRAL YRAVMLENYRNLEFVDSSLKSMMEFSSTRHSITGE 253 ZN426_HUMAN EKTPAGRIVADCLTDCYQDSVTFDDVAVDFTQEEWTLLDSTQRSL YSDVMLENYKNLATVGGQIIKPSLISWLEQEESRT 254 ZN674_HUMAN AMSQESLTFKDVFVDFTLEEWQQLDSAQKNLYRDVMLENYSHLVS VGHLVGKPDVIFRLGPGDESWMADGGTPVRTCAGE 255 ZN627_HUMAN DSVAFEDVAVNFTLEEWALLDPSQKNLYRDVMRETERNLASVGKQ WEDQNIEDPFKIPRRNISHIPERLCESKEGGQGEE 256 ZNF20_HUMAN MFQDSVAFEDVAVSFTQEEWALLDPSQKNLYRDVMQETEKNLTSV GKTWKVQNIEDEYKNPRRNLSLMREKLCESKESHH 257 Z587B_HUMAN AVVATLRLSAQGTVTFEDVAVKFTQEEWNLLSEAQRCLYRDVTLE NLALMSSLGCWCGVEDEAAPSKQSIYIQRETQVRT 258 ZN316_HUMAN EEEEEDEDEDDLLTAGCQELVTFEDVAVYFSLEEWERLEADQRGL YQEVMQENYGILVSLGYPIPKPDLIFRLEQGEEPW 259 ZN233_HUMAN TKFQEMVTFKDVAVVFTREELGLLDLAQRKLYQDVMLENERNLLS VGYQPFKLDVILQLGKEDKLRMMETEIQGDGCSGH 260 ZN611_HUMAN EEAAQKRKGKEPGMALPQGRLTERDVAIEFSLAEWKCLNPSQRAL YREVMLENYRNLEAVDISSKCMMKEVLSTGQGNTE 261 ZN556_HUMAN DTVVFEDVVVDFTLEEWALLNPAQRKLYRDVMLETFKHLASVDNE AQLKASGSISQQDTSGEKLSLKQKIEKFTRKNIWA 262 ZN234_HUMAN TTFKEGLTFKDVAVVFTEEELGLLDPVQRNLYQDVMLENERNLLS VGHHPFKHDVELLEKEKKLDIMKTATQRKGKSADK 263 ZN560_HUMAN SALQQEFWKIQTSNGIQMDLVTEDSVAVEFTQEEWTLLDPAQRNL YSDVMLENYKNLSSVGYQLFKPSLISWLEEEEELS 264 ZNF77_HUMAN DCVIFEEVAVNFTPEEWALLDHAQRSLYRDVMLETCRNLASLDCY IYVRTSGSSSQRDVFGNGISNDEEIVKFTGSDSWS 265 ZN682_HUMAN ELLTFRDVTIEFSLEEWEFLNPAQQSLYRKVMLENYRNLVSLGLT VSKPELISRLEQRQEPWNVKRHETIAKPPAMSSHY 266 ZN614_HUMAN IKTQESLTLEDVAVEFSWEEWQLLDTAQKNLYRDVMVENYNHLVS LGYQTSKPDVLSKLAHGQEPWTTDAKIQNKNCPGI 267 ZN785_HUMAN PAHVPGEAGPRRTRESRPGAVSFADVAVYFSPEEWECLRPAQRAL YRDVMRETFGHLGALGFSVPKPAFISWVEGEVEAW 268 ZN445_HUMAN GCPGDQVTPTRSLTAQLQETMTFKDVEVTFSQDEWGWLDSAQRNL YRDVMLENYRNMASLVGPFTKPALISWLEAREPWG 269 ZFP30_HUMAN ARDLVMFRDVAVDESQEEWECLNSYQRNLYRDVILENYSNLVSLA GCSISKPDVITLLEQGKEPWMVVRDEKRRWTLDLE 270 ZN225_HUMAN TTLKEAVTFKDVAVVFTEEELRLLDLAQRKLYREVMLENERNLLS VGHQSLHRDTFHFLKEEKFWMMETATQREGNLGGK 271 ZN551_HUMAN SPPSPRSSMAAVALRDSAQGMTFEDVAIYFSQEEWELLDESQREL YCDVMLENFAHVTSLGYCHGMENEAIASEQSVSIQ 272 ZN610_HUMAN DEEAQKRKAKESGMALPQGRLTEMDVAIEFSQEEWKSLDPGQRAL YRDVMLENYRNLVELGICLPDLSIISMLKQRREPL 273 ZN528_HUMAN ALTQGPLKEMDVAIEFSQEEWKCLDPAQRTLYRDVMLENYRNLVS LGICLPDLSVTSMLEQKRDPWTLQSEEKIANDPDG 274 ZN284_HUMAN TMFKEAVTFKDVAVVFTEEELGLLDVSQRKLYRDVMLENERNLLS VGHQLSHRDTFHFQREEKFWIMETATQREGNSGGK 275 ZN418_HUMAN QGTVAFEDVAVNESQEEWSLLSEVQRCLYHDVMLENWVLISSLGC WCGSEDEEAPSKKSISIQRVSQVSTPGAGVSPKKA 276 MPP8_HUMAN AEAFGDSEEDGEDVFEVEKILDMKTEGGKVLYKVRWKGYTSDDDT WEPEIHLEDCKEVLLEFRKKIAENKAKAVRKDIQR 277 ZN490_HUMAN VLQMQNSEHHGQSIKTQTDSISLEDVAVNFTLEEWALLDPGQRNI YRDVMRATFKNLACIGEKWKDQDIEDEHKNQGRNL 278 ZN805_HUMAN AMALTDPAQVSVTFDDVAVTFTQEEWGQLDLAQRTLYQEVMLENC GLLVSLGCPVPRPELIYHLEHGQEPWTRKEDLSQG 279 Z780B_HUMAN VHGSVTFRDVAIDFSQEEWECLQPDQRTLYRDVMLENYSHLISLG SSISKPDVITLLEQEKEPWIVVSKETSRWYPDLES 280 ZN763_HUMAN DPVACEDVAVNFTQEEWALLDISQRKLYREVMLETERNLTSIGKK WKDQNIEYEYQNPRRNERSLIEGNVNEIKEDSHCG 281 ZN285_HUMAN IKFQERVTFKDVAVVFTKEELALLDKAQINLYQDVMLENERNLML VRDGIKNNILNLQAKGLSYLSQEVLHCWQIWKQRI 282 ZNF85_HUMAN GPLTERDVAIEFSLKEWQCLDTAQRNLYRNVMLENYRNLVELGIT VSKPDLITCLEQGKEAWSMKRHEIMVAKPTVMCSH 283 ZN223_HUMAN TMSKEAVTFKDVAVVFTEEELGLLDLAQRKLYRDVMLENERNLLS VGHQPFHRDTFHFLREEKFWMMDIATQREGNSGGK 284 ZNF90_HUMAN GPLEFRDVAIEFSLEEWHCLDTAQQNLYRDVMLENYRHLVELGIV VTKPDLITCLEQGKKPFTVKRHEMIAKSPVMCFHF 285 ZN557_HUMAN GHTEGGELVNELLKSWLKGLVTFEDVAVEFTQEEWALLDPAQRTL YRDVMLENCRNLASLGNQVDKPRLISQLEQEDKVM 286 ZN425_HUMAN AEPASVTVTEDDVALYFSEQEWEILEKWQKQMYKQEMKTNYETLD SLGYAFSKPDLITWMEQGRMLLISEQGCLDKTRRT 287 ZN229_HUMAN HSQASAISQDREEKIMSQEPLSFKDVAVVFTEEELELLDSTQRQL YQDVMQENFRNLLSVGERNPLGDKNGKDTEYIQDE 288 ZN606_HUMAN GSLEEGRRATGLPAAQVQEPVTFKDVAVDFTQEEWGQLDLVQRTL YRDVMLETYGHLLSVGNQIAKPEVISLLEQGEEPW 289 ZN155_HUMAN TTFKEAVTFKDVAVVFTEEELGLLDPAQRKLYRDVMLENERNLLS VGHQPFHQDTCHFLREEKFWMMGTATQREGNSGGK 290 ZN222_HUMAN AKLYEAVTFKDVAVIFTEEELGLLDPAQRKLYRDVMLENERNLLS VGGKIQTEMETVPEAGTHEEFSCKQIWEQIASDLT 291 ZN442_HUMAN RSDLFLPDSQTNEERKQYDSVAFEDVAVNFTQEEWALLGPSQKSL YRDVMWETIRNLDCIGMKWEDTNIEDQHRNPRRSL 292 ZNF91_HUMAN PGTPGSLEMGLLTFRDVAIEFSPEEWQCLDTAQQNLYRNVMLENY RNLAFLGIALSKPDLITYLEQGKEPWNMKQHEMVD 293 ZN135_HUMAN TPGVRVSTDPEQVTFEDVVVGFSQEEWGQLKPAQRTLYRDVMLDT FRLLVSVGHWLPKPNVISLLEQEAELWAVESRLPQ 294 ZN778_HUMAN EQTQAAGMVAGWLINCYQDAVTEDDVAVDFTQEEWTLLDPSQRDL YRDVMLENYENLASVEWRLKTKGPALRQDRSWFRA 295 RYBP_HUMAN PSEANSIQSANATTKTSETNHTSRPRLKNVDRSTAQQLAVTVGNV TVIITDFKEKTRSSSTSSSTVTSSAGSEQQNQSSS 296 ZN534_HUMAN ALTQGQLSFSDVAIEFSQEEWKCLDPGQKALYRDVMLENYRNLVS LGEDNVRPEACICSGICLPDLSVTSMLEQKRDPWT 297 ZN586_HUMAN AAAAALRAPAQSSVTFEDVAVNESLEEWSLLNEAQRCLYRDVMLE TLTLISSLGCWHGGEDEAAPSKQSTCIHIYKDQGG 298 ZN567_HUMAN AQGSVSFNDVTVDFTQEEWQHLDHAQKTLYMDVMLENYCHLISVG CHMTKPDVILKLERGEEPWTSFAGHTCLEENWKAE 299 ZN440_HUMAN DPVAFKDVAVNFTQEEWALLDISQRKLYREVMLETERNLTSLGKR WKDQNIEYEHQNPRRNERSLIEEKVNEIKDDSHCG 300 ZN583_HUMAN SKDLVTFGDVAVNFSQEEWEWLNPAQRNLYRKVMLENYRSLVSLG VSVSKPDVISLLEQGKEPWMVKKEGTRGPCPDWEY 301 ZN441_HUMAN DSVAFEDVAINFTCEEWALLGPSQKSLYRDVMQETIRNLDCIGMI WQNHDIEEDQYKDLRRNLRCHMVERACEIKDNSQC 302 ZNF43_HUMAN GPLTFMDVAIEFCLEEWQCLDIAQQNLYRNVMLENYRNLVELGIA VSKPDLITCLEQEKEPWEPMRRHEMVAKPPVMCSH 303 CBX5_HUMAN QSNDIARGFERGLEPEKIIGATDSCGDLMFLMKWKDTDEADLVLA KEANVKCPQIVIAFYEERLTWHAYPEDAENKEKET 304 ZN589_HUMAN ALPAKDSAWPWEEKPRYLGPVTFEDVAVLFTEAEWKRLSLEQRNL YKEVMLENLRNLVSLAESKPEVHTCPSCPLAFGSQ 305 ZNF10_HUMAN DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLEN YKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQ 306 ZN563_HUMAN DAVAFEDVAVNFTQEEWALLGPSQKNLYRYVMQETIRNLDCIRMI WEEQNTEDQYKNPRRNLRCHMVERFSESKDSSQCG 307 ZN561_HUMAN EKTKVERMVEDYLASGYQDSVTEDDVAVDETPEEWALLDTTEKYL YRDVMLENYMNLASVEWEIQPRTKRSSLQQGELKN 308 ZN136_HUMAN DSVAFEDVDVNFTQEEWALLDPSQKNLYRDVMWETMRNLASIGKK WKDQNIKDHYKHRGRNLRSHMLERLYQTKDGSQRG 309 ZN630_HUMAN IESQEPVTFEDVAVDFTQEEWQQLNPAQKTLHRDVMLETYNHLVS VGCSGIKPDVIFKLEHGKDPWIIESELSRWIYPDR 310 ZN527_HUMAN AVGLCKAMSQGLVTERDVALDESQEEWEWLKPSQKDLYRDVMLEN YRNLVWLGLSISKPNMISLLEQGKEPWMVERKMSQ 311 ZN333_HUMAN DKVEEEAMAPGLPTACSQEPVTFADVAVVFTPEEWVELDSTQRSL YRDVMLENYRNLASVADQLCKPNALSYLEERGEQW 312 Z324B_HUMAN TFEDVAVYFSQEEWGLLDTAQRALYRHVMLENFTLVTSLGLSTSR PRVVIQLERGEEPWVPSGKDMTLARNTYGRLNSGS 313 ZN786_HUMAN AEPPRLPLTFEDVAIYFSEQEWQDLEAWQKELYKHVMRSNYETLV SLDDGLPKPELISWIEHGGEPERKWRESQKSGNII 314 ZN709_HUMAN DSVVFEDVAVNFTQEEWALLGPSQKKLYRDVMQETFVNLASIGEN WEEKNIEDHKNQGRKLRSHMVERLCERKEGSQFGE 315 ZN792_HUMAN AAAALRDPAQGCVTFEDVTIYFSQEEWVLLDEAQRLLYCDVMLEN FALIASLGLISFRSHIVSQLEMGKEPWVPDSVDMT 316 ZN599_HUMAN AAPALALVSFEDVVVTFTGEEWGHLDLAQRTLYQEVMLETCRLLV SLGHPVPKPELIYLLEHGQELWTVKRGLSQSTCAG 317 ZN613_HUMAN IKSQESLTLEDVAVEFTWEEWQLLGPAQKDLYRDVMLENYSNLVS VGYQASKPDALFKLEQGEPWTVENEIHSQICPEIK 318 ZF69B_HUMAN GESLESRVTLGSLTAESQELLTFKDVSVDFTQEEWGQLAPAHRNL YREVMLENYGNLVSVGCQLSKPGVISQLEKGEEPW 319 ZN799_HUMAN ASVALEDVAVNFTREEWALLGPCQKNLYKDVMQETIRNLDCVGMK WKDQNIEDQYRYPRKNLRCRMLERFVESKDGTQCG 320 ZN569_HUMAN TESQGTVTFKDVAIDFTQEEWKRLDPAQRKLYRNVMLENYNNLIT VGYPFTKPDVIFKLEQEEEPWVMEEEVLRRHWQGE 321 ZN564_HUMAN DSVASEDVAVNFTLEEWALLDPSQKKLYRDVMRETERNLACVGKK WEDQSIEDWYKNQGRILRNHMEEGLSESKEYDQCG 322 ZN546_HUMAN EETQGELTSSCGSKTMANVSLAFRDVSIDLSQEEWECLDAVQRDL YKDVMLENYSNLVSLGYTIPKPDVITLLEQEKEPW 323 ZFP92_HUMAN AAILLTTRPKVPVSFEDVSVYFTKTEWKLLDLRQKVLYKRVMLEN YSHLVSLGFSFSKPHLISQLERGEGPWVADIPRTW 324 YAF2_HUMAN KDKVEKEKSEKETTSKKNSHKKTRPRLKNVDRSSAQHLEVTVGDL TVIITDFKEKTKSPPASSAASADQHSQSGSSSDNT 325 ZN723_HUMAN GPLTFTDVAIKFSLEEWQFLDTAQQNLYRDVMLENYRNLVELGVG VSKPDLITCLEQGKEPWNMKRHKMVAKPPVVCSHF 326 ZNF34_HUMAN RKPNPQAMAALFLSAPPQAEVTFEDVAVYLSREEWGRLGPAQRGL YRDVMLETYGNLVSLGVGPAGPKPGVISQLERGDE 327 ZN439_HUMAN LSLSPILLYTCEMFQDPVAFKDVAVNFTQEEWALLDISQKNLYRE VMLETFWNLTSIGKKWKDQNIEYEYQNPRRNERSV 328 ZFP57_HUMAN AAGEPRSLLFFQKPVTFEDVAVNETQEEWDCLDASQRVLYQDVMS ETFKNLTSVARIFLHKPELITKLEQEEEQWRETRV 329 ZNF19_HUMAN AAMPLKAQYQEMVTFEDVAVHFTKTEWTGLSPAQRALYRSVMLEN FGNLTALGYPVPKPALISLLERGDMAWGLEAQDDP 330 ZN404_HUMAN ARVPLTFSDVAIDFSQEEWEYLNSDQRDLYRDVMLENYTNLVSLD FNFTTESNKLSSEKRNYEVNAYHQETWKRNKTENL 331 ZN274_HUMAN ASRLPTAWSCEPVTFEDVTLGFTPEEWGLLDLKQKSLYREVMLEN YRNLVSVEHQLSKPDVVSQLEEAEDEWPVERGIPQ 332 CBX3_HUMAN SKKKRDAADKPRGFARGLDPERIIGATDSSGELMFLMKWKDSDEA DLVLAKEANMKCPQIVIAFYEERLTWHSCPEDEAQ 333 ZNF30_HUMAN AHKYVGLQYHGSVTFEDVAIAFSQQEWESLDSSQRGLYRDVMLEN YRNLVSMGHSRSKPHVIALLEQWKEPEVTVRKDGR 334 ZN250_HUMAN AAARLLPVPAGPQPLSFQAKLTFEDVAVLLSQDEWDRLCPAQRGL YRNVMMETYGNVVSLGLPGSKPDIISQLERGEDPW 335 ZN570_HUMAN AVGLLKAMYQELVTERDVAVDESQEEWDCLDSSQRHLYSNVMLEN YRILVSLGLCFSKPSVILLLEQGKAPWMVKRELTK 336 ZN675_HUMAN GLLTFRDVAIEFSLEEWQCLDTAQRNLYKNVILENYRNLVELGIA VSKQDLITCLEQEKEPLTVKRHEMVNEPPVMCSHE 337 ZN695_HUMAN GLLAFRDVALEFSPEEWECLDPAQRSLYRDVMLENYRNLISLGED SENMQFLFHSLAMSKPELIICLEARKEPWNVNTEK 338 ZN548_HUMAN NLTEGRVVFEDVAIYFSQEEWGHLDEAQRLLYRDVMLENLALLSS LGSWHGAEDEEAPSQQGFSVGVSEVTASKPCLSSQ 339 ZN132_HUMAN GPAQHTSWPCGSAVPTLKSMVTFEDVAVYFSQEEWELLDAAQRHL YHSVMLENLELVTSLGSWHGVEGEGAHPKQNVSVE 340 ZN738_HUMAN SGYPGAERNLLEYSYFEKGPLTFRDVVIEFSQEEWQCLDTAQQDL YRKVMLENFRNLVELGIDVSKPDLITCLEQGKDPW 341 ZN420_HUMAN ARKLVMERDVAIDFSQEEWECLDSAQRDLYRDVMLENYSNLVSLD LPSRCASKDLSPEKNTYETELSQWEMSDRLENCDL 342 ZN626_HUMAN GPLQFRDVAIEFSLEEWHCLDTAQRNLYRNVMLENYSNLVELGIT VSKPDLITCLEQGRKPLTMKRNEMIAKPSVMCSHE 343 ZN559_HUMAN VAGWLTNYSQDSVTFEDVAVDFTQEEWTLLDQTQRNLYRDVMLEN YKNLVAVDWESHINTKWSAPQQNFLQGKTSSVVEM 344 ZN460_HUMAN AAAWMAPAQESVTFEDVAVTFTQEEWGQLDVTQRALYVEVMLETC GLLVALGDSTKPETVEPIPSHLALPEEVSLQEQLA 345 ZN268_HUMAN VLEWLFISQEQPKITKSWGPLSEMDVFVDFTWEEWQLLDPAQKCL YRSVMLENYSNLVSLGYQHTKPDIIFKLEQGEELC 346 ZN304_HUMAN AAAVLMDRVQSCVTFEDVEVYFSREEWELLEEAQRFLYRDVMLEN FALVATLGFWCEAEHEAPSEQSVSVEGVSQVRTAE 347 ZIM2_HUMAN AGSQFPDFKHLGTFLVFEELVTFEDVLVDESPEELSSLSAAQRNL YREVMLENYRNLVSLGHQFSKPDIISRLEEEESYA 348 ZN605_HUMAN IQSQISFEDVAVDETLEEWQLLNPTQKNLYRDVMLENYSNLVFLE VWLDNPKMWLRDNQDNLKSMERGHKYDVEGKIENS 349 ZN844_HUMAN DLVAFEDVAVNFTQEEWSLLDPSQKNLYREVMQETLRNLASIGEK WKDQNIEDQYKNPRNNLRSLLGERVDENTEENHCG 350 SUMO5_HUMAN KDEDIKLRVIGQDSSEIHFKVKMTTPLKKLKKSYCQRQGVPVNSL RFLFEGQRIADNHTPEELGMEEEDVIEVYQEQIGG 351 ZN101_HUMAN DSVAFEDVAVNFTQEEWALLSPSQKNLYRDVTLETERNLASVGIQ WKDQDIENLYQNLGIKLRSLVERLCGRKEGNEHRE 352 ZN783_HUMAN RNFWILRLPPGSKGEAPKVPVTEDDVAVYFSELEWGKLEDWQKEL YKHVMRGNYETLVSLDYAISKPDILTRIERGEEPC 353 ZN417_HUMAN AAAAPRRPTQQGTVTFEDVAVNFSQEEWCLLSEAQRCLYRDVMLE NLALISSLGCWCGSKDEEAPCKQRISVQRESQSRT 354 ZN182_HUMAN SGEDSGSFYSWQKAKREQGLVTFEDVAVDETQEEWQYLNPPQRTL YRDVMLETYSNLVFVGQQVTKPNLILKLEVEECPA 355 ZN823_HUMAN DSVAFEDVAVNFTQEEWALLGPSQKSLYRNVMQETIRNLDCIEMK WEDQNIGDQCQNAKRNLRSHTCEIKDDSQCGETFG 356 ZN177_HUMAN AAGWLTTWSQNSVTFQEVAVDFSQEEWALLDPAQKNLYKDVMLEN FRNLASVGYQLCRHSLISKVDQEQLKTDERGILQG 357 ZN197_HUMAN ENPRNQLMALMLLTAQPQELVMFEEVSVCFTSEEWACLGPIQRAL YWDVMLENYGNVTSLEWETMTENEEVTSKPSSSQR 358 ZN717_HUMAN LETYNSLVSLQELVSFEEVAVHFTWEEWQDLDDAQRTLYRDVMLE TYSSLVSLGHCITKPEMIFKLEQGAEPWIVEETPN 359 ZN669_HUMAN RHFRRPEPCREPLASPIQDSVAFEDVAVNFTQEEWALLDSSQKNL YREVMQETCRNLASVGSQWKDQNIEDHFEKPGKDI 360 ZN256_HUMAN AAAELTAPAQGIVTFEDVAVYFSWKEWGLLDEAQKCLYHDVMLEN LTLTTSLGGSGAGDEEAPYQQSTSPQRVSQVRIPK 36 ZN251_HUMAN AATFQLPGHQEMPLTFQDVAVYFSQAEGRQLGPQQRALYRDVMLE NYGNVASLGFPVPKPELISQLEQGKELWVLNLLGA 362 CBX4_HUMAN RSEAGEPPSSLQVKPETPASAAVAVAAAAAPTTTAEKPPAEAQDE PAESLSEFKPFFGNIIITDVTANCLTVTFKEYVTV 363 PCGF2_HUMAN HRTTRIKITELNPHLMCALCGGYFIDATTIVECLHSFCKTCIVRY LETNKYCPMCDVQVHKTRPLLSIRSDKTLQDIVYK 364 CDY2_HUMAN ASQEFEVEAIVDKRQDKNGNTQYLVRWKGYDKQDDTWEPEQHLMN CEKCVHDENRRQTEKQKKLTWTTTSRIFSNNARRR 365 CDYL2_HUMAN ASGDLYEVERIVDKRKNKKGKWEYLIRWKGYGSTEDTWEPEHHLL HCEEFIDEFNGLHMSKDKRIKSGKQSSTSKLLRDS 366 HERC2_HUMAN TLIRKADLENHNKDGGFWTVIDGKVYDIKDFQTQSLTGNSILAQF AGEDPVVALEAALQFEDTRESMHAFCVGQYLEPDQ 367 ZN562_HUMAN EKTKIGTMVEDHRSNSYQDSVTFDDVAVEFTPEEWALLDTTQKYL YRDVMLENYMNLASVDFFFCLTSEWEIQPRTKRSS 368 ZN461_HUMAN AHELVMFRDVAIDVSQEEWECLNPAQRNLYKEVMLENYSNLVSLG LSVSKPAVISSLEQGKEPWMVVREETGRWCPGTWK 369 Z324A_HUMAN AFEDVAVYFSQEEWGLLDTAQRALYRRVMLDNFALVASLGLSTSR PRVVIQLERGEEPWVPSGTDTTLSRTTYRRRNPGS 370 ZN766_HUMAN AQLRRGHLTFRDVAIEFSQEEWKCLDPVQKALYRDVMLENYRNLV SLGICLPDLSIISMMKQRTEPWTVENEMKVAKNPD 371 ID2_HUMAN SDHSLGISRSKTPVDDPMSLLYNMNDCYSKLKELVPSIPQNKKVS KMEILQHVIDYILDLQIALDSHPTIVSLHHQRPGQ 372 TOX_HUMAN KDPNEPQKPVSAYALFERDTQAAIKGQNPNATFGEVSKIVASMWD GLGEEQKQVYKKKTEAAKKEYLKQLAAYRASLVSK 373 ZN274_HUMAN QEEKQEDAAICPVTVLPEEPVTFQDVAVDESREEWGLLGPTQRTE YRDVMLETFGHLVSVGWETTLENKELAPNSDIPEE 374 SCMH1_HUMAN DASRLSGRDPSSWTVEDVMQFVREADPQLGPHADLERKHEIDGKA LLLLRSDMMMKYMGLKLGPALKLSYHIDRLKQGKE 375 ZN214_HUMAN AVTFEDVTIIFTWEEWKFLDSSQKRLYREVMWENYTNVMSVENWN ESYKSQEEKFRYLEYENFSYWQGWWNAGAQMYENQ 376 CBX7_HUMAN ELSAIGEQVFAVESIRKKRVRKGKVEYLVKWKGWPPKYSTWEPEE HILDPRLVMAYEEKEERDRASGYRKRGPKPKRLLL 377 ID1_HUMAN GGAGARLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSK VEILQHVIDYIRDLQLELNSESEVGTPGGRGLPVR 378 CREM_HUMAN VVMAASPGSLHSPQQLAEEATRKRELRLMKNREAAKECRRRKKEY VKCLESRVAVLEVQNKKLIEELETLKDICSPKTDY 379 SCX_HUMAN GGGPGGRPGREPRQRHTANARERDRTNSVNTAFTALRTLIPTEPA DRKLSKIETLRLASSYISHLGNVLLAGEACGDGQP 380 ASCLI_HUMAN SGFGYSLPQQQPAAVARRNERERNRVKLVNLGFATLREHVPNGAA NKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQ 381 ZN764_HUMAN APLPPRDPNGAGPEWREPGAVSFADVAVYFCREEWGCLRPAQRAL YRDVMRETYGHLSALGIGGNKPALISWVEEEAELW 382 SCML2_HUMAN KQGFSKDPSTWSVDEVIQFMKHTDPQISGPLADLERQHEIDGKAL FLLKSDVMMKYMGLKLGPALKLCYYIEKLKEGKYS 383 TWSTI_HUMAN SGGGSPQSYEELQTQRVMANVRERQRTQSLNEAFAALRKIIPTLP SDKLSKIQTLKLAARYIDFLYQVLQSDELDSKMAS 384 CREB1_HUMAN IAPGVVMASSPALPTQPAEEAARKREVRLMKNREAARECRRKKKE YVKCLENRVAVLENQNKTLIEELKALKDLYCHKSD 385 TERFI_HUMAN SRIPVSKSQPVTPEKHRARKRQAWLWEEDKNLRSGVRKYGEGNWS KILLHYKENNRTSVMLKDRWRTMKKLKLISSDSED 386 ID3_HUMAN SLAIARGRGKGPAAEEPLSLLDDMNHCYSRLRELVPGVPRGTQLS QVEILQRVIDYILDLQVVLAEPAPGPPDGPHLPIQ 387 CBX8_HUMAN GSGPPSSGGGLYRDMGAQGGRPSLIARIPVARILGDPEEESWSPS LTNLEKVVVTDVTSNFLTVTIKESNTDQGFFKEKR 388 CBX4_HUMAN ELPAVGEHVFAVESIEKKRIRKGRVEYLVKWRGWSPKYNTWEPEE NILDPRLLIAFQNRERQEQLMGYRKRGPKPKPLVV 389 GSX1_HUMAN VDSSSNQLPSSKRMRTAFTSTQLLELEREFASNMYLSRLRRIEIA TYLNLSEKQVKIWFQNRRVKHKKEGKGSNHRGGGG 390 NKX22_HUMAN TPGGGGDAGKKRKRRVLFSKAQTYELERRFRQQRYLSAPEREHLA SLIRLTPTQVKIWFQNHRYKMKRARAEKGMEVTPL 391 ATF1_HUMAN QTVVMTSPVTLTSQTTKTDDPQLKREIRLMKNREAARECRRKKKE YVKCLENRVAVLENQNKTLIEELKTLKDLYSNKSV 392 TWST2_HUMAN KGSPSAQSFEELQSQRILANVRERQRTQSLNEAFAALRKIIPTLP SDKLSKIQTLKLAARYIDFLYQVLQSDEMDNKMTS 393 ZNF17_HUMAN NLTEDYMVFEDVAIHFSQEEWGILNDVQRHLHSDVMLENFALLSS VGCWHGAKDEEAPSKQCVSVGVSQVTTLKPALSTQ 394 TOX3_HUMAN KDPNEPQKPVSAYALFFRDTQAAIKGQNPNATFGEVSKIVASMWD SLGEEQKQVYKRKTEAAKKEYLKALAAYRASLVSK 395 TOX4_HUMAN KDPNEPQKPVSAYALFERDTQAAIKGQNPNATFGEVSKIVASMWD SLGEEQKQVYKRKTEAAKKEYLKALAAYKDNQECQ 396 ZMYM3_HUMAN LDGSTWDFCSEDCKSKYLLWYCKAARCHACKRQGKLLETIHWRGQ IRHFCNQQCLLRFYSQQNQPNLDTQSGPESLLNSQ 397 I2BP1_HUMAN ASVQASRRQWCYLCDLPKMPWAMVWDESEAVCRGCVNFEGADRIE LLIDAARQLKRSHVLPEGRSPGPPALKHPATKDLA 398 RHXF1_HUMAN MEGPQPENMQPRTRRTKFTLLQVEELESVFRHTQYPDVPTRRELA ENLGVTEDKVRVWFKNKRARCRRHQRELMLANELR 399 SSX2_HUMAN PKIMPKKPAEEGNDSEEVPEASGPQNDGKELCPPGKPTTSEKIHE RSGPKRGEHAWTHRLRERKQLVIYEEISDPEEDDE 400 I2BPL_HUMAN SAAQVSSSRRQSCYLCDLPRMPWAMIWDESEPVCRGCVNYEGADR IEFVIETARQLKRAHGCFQDGRSPGPPPPVGVKTV 401 ZN680_HUMAN PGPPGSLEMGPLTERDVAIEFSLEEWQCLDTAQRNLYRKVMFENY RNLVFLGIAVSKPHLITCLEQGKEPWNRKRQEMVA 402 CBX1_HUMAN NKKKVEEVLEEEEEEYVVEKVLDRRVVKGKVEYLLKWKGESDEDN TWEPEENLDCPDLIAEFLQSQKTAHETDKSEGGKR 403 TRI68_HUMAN LANVVEKVRLLRLHPGMGLKGDLCERHGEKLKMFCKEDVLIMCEA CSQSPEHEAHSVVPMEDVAWEYKWELHEALEHLKK 404 HXA13_HUMAN VVSHPSDASSYRRGRKKRVPYTKVQLKELEREYATNKFITKDKRR RISATTNLSERQVTIWFQNRRVKEKKVINKLKTTS 405 PHC3_HUMAN ENSDLLPVAQTEPSIWTVDDVWAFIHSLPGCQDIADEFRAQEIDG QALLLLKEDHLMSAMNIKLGPALKICARINSLKES 406 TCF24_HUMAN AGPGGGSRSGSGRPAAANAARERSRVQTLRHAFLELQRTLPSVPP DTKLSKLDVLLLATTYIAHLTRSLQDDAEAPADAG 407 CBX3_HUMAN QNGKSKKVEEAEPEEFVVEKVLDRRVVNGKVEYFLKWKGFTDADN TWEPEENLDCPELIEAFLNSQKAGKEKDGTKRKSL 408 HXB13_HUMAN QHPPDACAFRRGRKKRIPYSKGQLRELEREYAANKFITKDKRRKI SAATSLSERQITIWFQNRRVKEKKVLAKVKNSATP 409 HEY1_HUMAN SMSPTTSSQILARKRRRGIIEKRRRDRINNSLSELRRLVPSAFEK QGSAKLEKAEILQMTVDHLKMLHTAGGKGYFDAHA 410 PHC2_HUMAN LVGMGHHELPSEPTKWNVEDVYEFIRSLPGCQEIAEEFRAQEIDG QALLLLKEDHLMSAMNIKLGPALKIYARISMLKDS 411 ZNF81_HUMAN PANEDAPQPGEHGSACEVSVSFEDVTVDESREEWQQLDSTQRRLY QDVMLENYSHLLSVGFEVPKPEVIFKLEQGEGPWT 412 FIGLA_HUMAN GYSSTENLQLVLERRRVANAKERERIKNLNRGFARLKALVPFLPQ SRKPSKVDILKGATEYIQVLSDLLEGAKDSKKQDP 413 SAM11_HUMAN EEAPAPEDVTKWTVDDVCSFVGGLSGCGEYTRVFREQGIDGETLP LLTEEHLLTNMGLKLGPALKIRAQVARRLGRVFYV 414 KMT2B_HUMAN GGTLAHTPRRSLPSHHGKKMRMARCGHCRGCLRVQDCGSCVNCLD KPKFGGPNTKKQCCVYRKCDKIEARKMERLAKKGR 415 HEY2_HUMAN LNSPTTTSQIMARKKRRGIIEKRRRDRINNSLSELRRLVPTAFEK QGSAKLEKAEILQMTVDHLKMLQATGGKGYFDAHA 416 JDP2_HUMAN QPVKSELDEEEERRKRRREKNKVAAARCRNKKKERTEFLQRESER LELMNAELKTQIEELKQERQQLILMLNRHRPTCIV 417 HXC13_HUMAN LQPEVSSYRRGRKKRVPYTKVQLKELEKEYAASKFITKEKRRRIS ATTNLSERQVTIWFQNRRVKEKKVVSKSKAPHLHS 418 ASCL4_HUMAN LPVPLDSAFEPAFLRKRNERERQRVRCVNEGYARLRDHLPRELAD KRLSKVETLRAAIDYIKHLQELLERQAWGLEGAAG 419 HHEX_HUMAN SPFLQRPLHKRKGGQVRESNDQTIELEKKFETQKYLSPPERKRLA KMLQLSERQVKTWFQNRRAKWRRLKQENPQSNKKE 420 HERC2_HUMAN IAIATGSLHCVCCTEDGEVYTWGDNDEGQLGDGTTNAIQRPRLVA ALQGKKVNRVACGSAHTLAWSTSKPASAGKLPAQV 421 GSX2_HUMAN GGSDASQVPNGKRMRTAFTSTQLLELEREFSSNMYLSRLRRIEIA TYLNLSEKQVKIWFQNRRVKHKKEGKGTQRNSHAG 422 BIN1_HUMAN RLDLPPGFMFKVQAQHDYTATDTDELQLKAGDVVLVIPFQNPEEQ DEGWLMGVKESDWNQHKELEKCRGVFPENFTERVP 423 ETV7_HUMAN GICKLPGRLRIQPALWSREDVLHWLRWAEQEYSLPCTAEHGFEMN GRALCILTKDDFRHRAPSSGDVLYELLQYIKTQRR 424 ASCL3_HUMAN PNYRGCEYSYGPAFTRKRNERERQRVKCVNEGYAQLRHHLPEEYL EKRLSKVETLRAAIKYINYLQSLLYPDKAETKNNP 425 PHC1_HUMAN LHGINPVFLSSNPSRWSVEEVYEFIASLQGCQEIAEEFRSQEIDG QALLLLKEEHLMSAMNIKLGPALKICAKINVLKET 426 OTP_HUMAN QAGQQQGQQKQKRHRTRFTPAQLNELERSFAKTHYPDIFMREELA LRIGLTESRVQVWFQNRRAKWKKRKKTTNVFRAPG 427 I2BP2_HUMAN AAAVAVAAASRRQSCYLCDLPRMPWAMIWDFTEPVCRGCVNYEGA DRVEFVIETARQLKRAHGCFPEGRSPPGAAASAAA 428 VGLL2_HUMAN FSSQTPASIKEEEGSPEKERPPEAEYINSRCVLFTYFQGDISSVV DEHFSRALSQPSSYSPSCTSSKAPRSSGPWRDCSF 429 HXA11_HUMAN DKAGGSSGQRTRKKRCPYTKYQIRELEREFFFSVYINKEKRLQLS RMLNLTDRQVKIWFQNRRMKEKKINRDRLQYYSAN 430 PDLI4_HUMAN GAPLSGLQGLPECTRCGHGIVGTIVKARDKLYHPECFMCSDCGLN LKQRGYFELDERLYCESHAKARVKPPEGYDVVAVY 431 ASCL2_HUMAN RRPATAETGGGAAAVARRNERERNRVKLVNLGFQALRQHVPHGGA SKKLSKVETLRSAVEYIRALQRLLAEHDAVRNALA 432 CDX4_HUMAN TVQVTGKTRTKEKYRVVYTDHQRLELEKEFHCNRYITIQRKSELA VNLGLSERQVKIWFQNRRAKERKMIKKKISQFENS 433 ZN860_HUMAN EEAAQKRKEKEPGMALPQGHLTERDVAIEFSLEEWKCLDPTQRAL YRAMMLENYRNLHSVDISSKCMMKKESSTAQGNTE 434 LMBL4_HUMAN DIRASQVARWTVDEVAEFVQSLLGCEEHAKCFKKEQIDGKAFLLL TQTDIVKVMKIKLGPALKIYNSILMERHSQELPEE 435 PDIP3_HUMAN LSPLEGTKMTVNNLHPRVTEEDIVELFCVCGALKRARLVHPGVAE VVFVKKDDAITAYKKYNNRCLDGQPMKCNLHMNGN 436 NKX25_HUMAN DNAERPRARRRRKPRVLESQAQVYELERRFKQQRYLSAPERDQLA SVLKLTSTQVKIWFQNRRYKCKRQRQDQTLELVGL 437 CEBPB_HUMAN SQVKSKAKKTVDKHSDEYKIRRERNNIAVRKSRDKAKMRNLETQH KVLELTAENERLQKKVEQLSRELSTLRNLFKQLPE 438 ISLI_HUMAN KRDYIRLYGIKCAKCSIGFSKNDFVMRARSKVYHIECFRCVACSR QLIPGDEFALREDGLFCRADHDVVERASLGAGDPL 439 CDX2_HUMAN SLGSQVKTRTKDKYRVVYTDHQRLELEKEFHYSRYITIRRKAELA ATLGLSERQVKIWFQNRRAKERKINKKKLQQQQQQ 440 PROP1_HUMAN QGGQRGRPHSRRRHRTTFSPVQLEQLESAFGRNQYPDIWARESLA RDTGLSEARIQVWFQNRRAKQRKQERSLLQPLAHL 441 SIN3B_HUMAN DALTYLDQVKIRFGSDPATYNGFLEIMKEFKSQSIDTPGVIRRVS QLFHEHPDLIVGFNAFLPLGYRIDIPKNGKLNIQS 442 SMBT1_HUMAN RLHLDSNPLKWSVADVVRFIRSTDCAPLARIFLDQEIDGQALLLL TLPTVQECMDLKLGPAIKLCHHIERIKFAFYEQFA 443 HXC11_HUMAN AKGAAPNAPRTRKKRCPYSKFQIRELEREFFENVYINKEKRLQLS RMLNLTDRQVKIWFQNRRMKEKKLSRDRLQYESGN 444 HXC10_HUMAN TTGNWLTAKSGRKKRCPYTKHQTLELEKEFLENMYLTRERRLEIS KTINLTDRQVKIWFQNRRMKLKKMNRENRIRELTS 445 PRS6A_HUMAN YLVSNVIELLDVDPNDQEEDGANIDLDSQRKGKCAVIKTSTRQTY FLPVIGLVDAEKLKPGDLVGVNKDSYLILETLPTE 446 VSX1_HUMAN KASPTLGKRKKRRHRTVFTAHQLEELEKAFSEAHYPDVYAREMLA VKTELPEDRIQVWFQNRRAKWRKREKRWGGSSVMA 447 NKX23_HUMAN EESERPKPRSRRKPRVLFSQAQVFELERRFKQQRYLSAPEREHLA SSLKLTSTQVKIWFQNRRYKCKRQRQDKSLELGAH 448 MTG16_HUMAN VVPGSRQEEVIDHKLTEREWAEEWKHLNNLLNCIMDMVEKTRRSL TVLRRCQEADREELNHWARRYSDAEDTKKGPAPAA 449 HMX3_HUMAN ESPEKKPACRKKKTRTVFSRSQVFQLESTEDMKRYLSSSERAGLA ASLHLTETQVKIWFQNRRNKWKRQLAAELEAANLS 450 HMX1_HUMAN RGGVGVGGGRKKKTRTVFSRSQVFQLESTEDLKRYLSSAERAGLA ASLQLTETQVKIWFQNRRNKWKRQLAAELEAASLS 451 KIF22_HUMAN ELLAHGRQKILDLLNEGSARDLRSLQRIGPKKAQLIVGWRELHGP FSQVEDLERVEGITGKQMESELKANILGLAAGQRC 452 CSTF2_HUMAN ESPYGETISPEDAPESISKAVASLPPEQMFELMKQMKLCVQNSPQ EARNMLLQNPQLAYALLQAQVVMRIVDPEIALKIL 453 CEBPE_HUMAN AGPLHKGKKAVNKDSLEYRLRRERNNIAVRKSRDKAKRRILETQQ KVLEYMAENERLRSRVEQLTQELDTLRNLFRQIPE 454 DLX2_HUMAN IRIVNGKPKKVRKPRTIYSSFQLAALQRRFQKTQYLALPERAELA ASLGLTQTQVKIWFQNRRSKFKKMWKSGEIPSEQH 455 ZMYM3_HUMAN TVYQFCSPSCWTKFQRTSPEGGIHLSCHYCHSLFSGKPEVLDWQD QVFQFCCRDCCEDEKRLRGVVSQCEHCRQEKLLHE 456 PPARG_HUMAN TMVDTEMPFWPTNFGISSVDLSVMEDHSHSEDIKPFTTVDESSIS TPHYEDIPFTRTDPVVADYKYDLKLQEYQSAIKVE 457 PRICI_HUMAN GRHHAELLKPRCSACDEIIFADECTEAEGRHWHMKHFCCLECETV LGGQRYIMKDGRPFCCGCFESLYAEYCETCGEHIG 458 UNC4_HUMAN DPDKESPGCKRRRTRTNFTGWQLEELEKAFNESHYPDVEMREALA LRLDLVESRVQVWFQNRRAKWRKKENTKKGPGRPA 459 BARX2_HUMAN TEQPTPRQKKPRRSRTIFTELQLMGLEKKFQKQKYLSTPDRLDLA QSLGLTQLQVKTWYQNRRMKWKKMVLKGGQEAPTK 460 ALX3_HUMAN SMELAKNKSKKRRNRTTESTFQLEELEKVFQKTHYPDVYAREQLA LRTDLTEARVQVWFQNRRAKWRKRERYGKIQEGRN 461 TCF15_HUMAN GGGGGAGPVVVVRQRQAANARERDRTQSVNTAFTALRTLIPTEPV DRKLSKIETVRLASSYIAHLANVLLLGDSADDGQP 462 TERA_HUMAN IDDTVEGITGNLFEVYLKPYFLEAYRPIRKGDIFLVRGGMRAVEF KVVETDPSPYCIVAPDTVIHCEGEPIKREDEEESL 463 VSX2_HUMAN SALNQTKKRKKRRHRTIFTSYQLEELEKAFNEAHYPDVYAREMLA MKTELPEDRIQVWFQNRRAKWRKREKCWGRSSVMA 464 HXD12_HUMAN DGLPWGAAPGRARKKRKPYTKQQIAELENEFLVNEFINRQKRKEL SNRLNLSDQQVKIWFQNRRMKKKRVVLREQALALY 465 CDX1_HUMAN GGGGSGKTRTKDKYRVVYTDHQRLELEKEFHYSRYITIRRKSELA ANLGLTERQVKIWFQNRRAKERKVNKKKQQQQQPP 466 TCF23_HUMAN TRAGGLALGRSEASPENAARERSRVRTLRQAFLALQAALPAVPPD TKLSKLDVLVLAASYIAHLTRTLGHELPGPAWPPF 467 ALX1_HUMAN KCDSNVSSSKKRRHRTTFTSLQLEELEKVFQKTHYPDVYVREQLA LRTELTEARVQVWFQNRRAKWRKRERYGQIQQAKS 468 HXA10_HUMAN NAANWLTAKSGRKKRCPYTKHQTLELEKEFLENMYLTRERRLEIS RSVHLTDRQVKIWFQNRRMKLKKMNRENRIRELTA 469 RX_HUMAN LSEEEQPKKKHRRNRTTFTTYQLHELERAFEKSHYPDVYSREELA GKVNLPEVRVQVWFQNRRAKWRRQEKLEVSSMKLQ 470 CXXC5_HUMAN HMAGLAEYPMQGELASAISSGKKKRKRCGMCAPCRRRINCEQCSS CRNRKTGHQICKFRKCEELKKKPSAALEKVMLPTG 471 SCML1_HUMAN SITKHPSTWSVEAVVLELKQTDPLALCPLVDLERSHEIDGKALLL LTSDVLLKHLGVKLGTAVKLCYYIDRLKQGKCFEN 472 NFIL3_HUMAN ACRRKREFIPDEKKDAMYWEKRRKNNEAAKRSREKRRLNDLVLEN KLIALGEENATLKAELLSLKLKFGLISSTAYAQEI 473 DLX6_HUMAN EIRFNGKGKKIRKPRTIYSSLQLQALNHRFQQTQYLALPERAELA ASLGLTQTQVKIWFQNKRSKFKKLLKQGSNPHESD 474 MTG8_HUMAN GLHGTRQEEMIDHRLTDREWAEEWKHLDHLLNCIMDMVEKTRRSL TVLRRCQEADREELNYWIRRYSDAEDLKKGGGSSS 475 CBX8_HUMAN ELSAVGERVFAAEALLKRRIRKGRMEYLVKWKGWSQKYSTWEPEE NILDARLLAAFEEREREMELYGPKKRGPKPKTELL 476 CEBPD_HUMAN AREKSAGKRGPDRGSPEYRQRRERNNIAVRKSRDKAKRRNQEMQQ KLVELSAENEKLHQRVEQLTRDLAGLRQFFKQLPS 477 SEC13_HUMAN SGGCDNLIKLWKEEEDGQWKEEQKLEAHSDWVRDVAWAPSIGLPT STIASCSQDGRVFIWTCDDASSNTWSPKLLHKEND 478 FIP1_HUMAN VKGVDLDAPGSINGVPLLEVDLDSFEDKPWRKPGADLSDYENYGE NEDTWKAYCEKQKRIRMGLEVIPVTSTINKITAED 479 ALX4_HUMAN KADSESNKGKKRRNRTTFTSYQLEELEKVFQKTHYPDVYAREQLA MRTDLTEARVQVWFQNRRAKWRKRERFGQMQQVRT 480 LHX3_HUMAN TAKQREAEATAKRPRTTITAKQLETLKSAYNTSPKPARHVREQLS SETGLDMRVVQVWFQNRRAKEKRLKKDAGRQRWGQ 481 PRIC2_HUMAN GRHHAECLKPRCAACDEIIFADECTEAEGRHWHMKHFCCFECETV LGGQRYIMKEGRPYCCHCFESLYAEYCDTCAQHIG 482 MAGI3_HUMAN IIGGDRPDEFLQVKNVLKDGPAAQDGKIAPGDVIVDINGNCVLGH THADVVQMFQLVPVNQYVNLTLCRGYPLPDDSEDP 483 NELL1_HUMAN CCPECDTRVTSQCLDQNGHKLYRSGDNWTHSCQQCRCLEGEVDCW PLTCPNLSCEYTAILEGECCPRCVSDPCLADNITY 484 PRRX1_HUMAN LNSEEKKKRKQRRNRTTFNSSQLQALERVFERTHYPDAFVREDLA RRVNLTEARVQVWFQNRRAKERRNERAMLANKNAS 485 MTG8R_HUMAN GLNGGYQDELVDHRLTEREWADEWKHLDHALNCIMEMVEKTRRSM AVLRRCQESDREELNYWKRRYNENTELRKTGTELV 486 RAX2_HUMAN GPGEEAPKKKHRRNRTTFTTYQLHQLERAFEASHYPDVYSREELA AKVHLPEVRVQVWFQNRRAKWRRQERLESGSGAVA 487 DLX3_HUMAN VRMVNGKPKKVRKPRTIYSSYQLAALQRRFQKAQYLALPERAELA AQLGLTQTQVKIWFQNRRSKFKKLYKNGEVPLEHS 488 DLX1_HUMAN EVRFNGKGKKIRKPRTIYSSLQLQALNRRFQQTQYLALPERAELA ASLGLTQTQVKIWFQNKRSKFKKLMKQGGAALEGS 489 NKX26_HUMAN GRSEQPKARQRRKPRVLFSQAQVLALERRFKQQRYLSAPEREHLA SALQLTSTQVKIWFQNRRYKCKRQRQDKSLELAGH 490 NABI_HUMAN LPRTLGELQLYRILQKANLLSYFDAFIQQGGDDVQQLCEAGEEEF LEIMALVGMASKPLHVRRLQKALRDWVTNPGLENQ 491 SAMD7_HUMAN NLSLDEDIQKWTVDDVHSFIRSLPGCSDYAQVEKDHAIDGETLPL LTEEHLRGTMGLKLGPALKIQSQVSQHVGSMFYKK 492 PITX3_HUMAN SPEDGSLKKKQRRQRTHFTSQQLQELEATFQRNRYPDMSTREEIA VWTNLTEARVRVWFKNRRAKWRKRERSQQAELCKG 493 WDR5_HUMAN SNLLVSASDDKTLKIWDVSSGKCLKTLKGHSNYVFCCNENPQSNL IVSGSFDESVRIWDVKTGKCLKTLPAHSDPVSAVH 494 MEOX2_HUMAN GNYKSEVNSKPRKERTAFTKEQIRELEAEFAHHNYLTRLRRYEIA VNLDLTERQVKVWFQNRRMKWKRVKGGQQGAAARE 495 NAB2_HUMAN LPRTLGELQLYRVLQRANLLSYYETFIQQGGDDVQQLCEAGEEEF LEIMALVGMATKPLHVRRLQKALREWATNPGLESQ 496 DHX8_HUMAN PEEPTIGDIYNGKVTSIMQFGCFVQLEGLRKRWEGLVHISELRRE GRVANVADVVSKGQRVKVKVLSFTGTKTSLSMKDV 497 FOXA2_HUMAN YAFNHPFSINNLMSSEQQHHHSHHHHQPHKMDLKAYEQVMHYPGY GSPMPGSLAMGPVTNKTGLDASPLAADTSYYQGVY 498 CBX6_HUMAN TAAAGPAPPTAPEPAGASSEPEAGDWRPEMSPCSNVVVTDVTSNL LTVTIKEFCNPEDFEKVAAGVAGAAGGGGSIGASK 499 EMX2_HUMAN FLLHNALARKPKRIRTAFSPSQLLRLEHAFEKNHYVVGAERKQLA HSLSLTETQVKVWFQNRRTKFKRQKLEEEGSDSQQ 500 CPSF6_HUMAN KRIALYIGNLTWWTTDEDLTEAVHSLGVNDILEIKFFENRANGQS KGFALVGVGSEASSKKLMDLLPKRELHGQNPVVTP 501 HXC12_HUMAN SGAPWYPINSRSRKKRKPYSKLQLAELEGEFLVNEFITRQRRREL SDRLNLSDQQVKIWFQNRRMKKKRLLLREQALSFF 502 KDM4B_HUMAN SDNLYPESITSRDCVQLGPPSEGELVELRWTDGNLYKAKFISSVT SHIYQVEFEDGSQLTVKRGDIFTLEEELPKRVRSR 503 LMBL3_HUMAN GIPASKVSKWSTDEVSEFIQSLPGCEEHGKVFKDEQIDGEAFLLM TQTDIVKIMSIKLGPALKIFNSILMFKAAEKNSHN 504 PHX2A_HUMAN EPSGLHEKRKQRRIRTTFTSAQLKELERVFAETHYPDIYTREELA LKIDLTEARVQVWFQNRRAKFRKQERAASAKGAAG 505 EMX1_HUMAN LLLHGPFARKPKRIRTAFSPSQLLRLERAFEKNHYVVGAERKQLA GSLSLSETQVKVWFQNRRTKYKRQKLEEEGPESEQ 506 NC2B_HUMAN SSGNDDDLTIPRAAINKMIKETLPNVRVANDARELVVNCCTEFIH LISSEANEICNKSEKKTISPEHVIQALESLGFGSY 507 DLX4_HUMAN ERRPQAPAKKLRKPRTIYSSLQLQHLNQRFQHTQYLALPERAQLA AQLGLTQTQVKIWFQNKRSKYKKLLKQNSGGQEGD 508 SRY_HUMAN NVQDRVKRPMNAFIVWSRDQRRKMALENPRMRNSEISKQLGYQWK MLTEAEKWPFFQEAQKLQAMHREKYPNYKYRPRRK 509 ZN777_HUMAN EITRLAVWAAVQAVERKLEAQAMRLLTLEGRTGTNEKKIADCEKT AVEFANHLESKWVVLGTLLQEYGLLQRRLENMENL 510 NELL1_HUMAN CEKDIDECSEGIIECHNHSRCVNLPGWYHCECRSGFHDDGTYSLS GESCIDIDECALRTHTCWNDSACINLAGGEDCLCP 511 ZN398_HUMAN AAISLWTVVAAVQAIERKVEIHSRRLLHLEGRTGTAEKKLASCEK TVTELGNQLEGKWAVLGTLLQEYGLLQRRLENLEN 512 GATA3_HUMAN GQNRPLIKPKRRLSAARRAGTSCANCQTTTTTLWRRNANGDPVCN ACGLYYKLHNINRPLTMKKEGIQTRNRKMSSKSKK 513 BSH_HUMAN HAELPGKHCRRRKARTVESDSQLSGLEKRFEIQRYLSTPERVELA TALSLSETQVKTWFQNRRMKHKKQLRKSQDEPKAP 514 SF3B4_HUMAN QDATVYVGGLDEKVSEPLLWELFLQAGPVVNTHMPKDRVTGQHQG YGFVEFLSEEDADYAIKIMNMIKLYGKPIRVNKAS 515 TEADI_HUMAN PIDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRN ELIARYIKLRTGKTRTRKQVSSHIQVLARRKSRDE 516 TEAD3_HUMAN GLDNDAEGVWSPDIEQSFQEALAIYPPCGRRKIILSDEGKMYGRN ELIARYIKLRTGKTRTRKQVSSHIQVLARKKVREY 517 RGAP1_HUMAN DSVGTPQSNGGMRLHDFVSKTVIKPESCVPCGKRIKFGKLSLKCR DCRVVSHPECRDRCPLPCIPTLIGTPVKIGEGMLA 518 PHF1_HUMAN SAPHSMTASSSSVSSPSPGLPRRSAPPSPLCRSLSPGTGGGVRGG VGYLSRGDPVRVLARRVRPDGSVQYLVEWGGGGIF 519 FOXA1_HUMAN GDPHYSFNHPESINNLMSSSEQQHKLDFKAYEQALQYSPYGSTLP ASLPLGSASVTTRSPIEPSALEPAYYQGVYSRPVL 520 GATA2_HUMAN GQNRPLIKPKRRLSAARRAGTCCANCQTTTTTLWRRNANGDPVCN ACGLYYKLHNVNRPLTMKKEGIQTRNRKMSNKSKK 521 FOX03_HUMAN DSLSGSSLYSTSANLPVMGHEKFPSDLDLDMENGSLECDMESIIR SELMDADGLDENFDSLISTQNVVGLNVGNFTGAKQ 522 ZN212_HUMAN TEISLWTVVAAIQAVEKKMESQAARLQSLEGRTGTAEKKLADCEK MAVEFGNQLEGKWAVLGTLLQEYGLLQRRLENVEN 523 IRX4_HUMAN MDSGTRRKNATRETTSTLKAWLQEHRKNPYPTKGEKIMLAIITKM TLTQVSTWFANARRRLKKENKMTWPPRNKCADEKR 524 ZBED6_HUMAN NIEKQIYLPSTRAKTSIVWHFFHVDPQYTWRAICNLCEKSVSRGK PGSHLGTSTLQRHLQARHSPHWTRANKFGVASGEE 525 LHX4_HUMAN AKQNDDSEAGAKRPRTTITAKQLETLKNAYKNSPKPARHVREQLS SETGLDMRVVQVWFQNRRAKEKRLKKDAGRHRWGQ 526 SIN3A_HUMAN DALSYLDQVKLQFGSQPQVYNDELDIMKEFKSQSIDTPGVISRVS QLFKGHPDLIMGENTFLPPGYKIEVQTNDMVNVTT 527 RBBP7_HUMAN DDHTVCLWDINAGPKEGKIVDAKAIFTGHSAVVEDVAWHLLHESL FGSVADDQKLMIWDTRSNTTSKPSHLVDAHTAEVN 528 NKX61_HUMAN GSILLDKDGKRKHTRPTFSGQQIFALEKTFEQTKYLAGPERARLA YSLGMTESQVKVWFQNRRTKWRKKHAAEMATAKKK 529 TRI68_HUMAN DPTALVEAIVEEVACPICMTELREPMSIDCGHSFCHSCLSGLWEI PGESQNWGYTCPLCRAPVQPRNLRPNWQLANVVEK 530 R51A1_HUMAN QSLPKKVSLSSDTTRKPLEIRSPSAESKKPKWVPPAASGGSRSSS SPLVVVSVKSPNQSLRLGLSRLARVKPLHPNATST 531 MB3L1_HUMAN AKSSQRKQRDCVNQCKSKPGLSTSIPLRMSSYTFKRPVTRITPHP GNEVRYHQWEESLEKPQQVCWQRRLQGLQAYSSAG 532 DLX5_HUMAN VRMVNGKPKKVRKPRTIYSSFQLAALQRRFQKTQYLALPERAELA ASLGLTQTQVKIWFQNKRSKIKKIMKNGEMPPEHS 533 NOTCI_HUMAN LQCNNHACGWDGGDCSLNENDPWKNCTQSLQCWKYFSDGHCDSQC NSAGCLEDGEDCQRAEGQCNPLYDQYCKDHFSDGH 534 TERF2_HUMAN ETWVEEDELFQVQAAPDEDSTTNITKKQKWTVEESEWVKAGVQKY GEGNWAAISKNYPFVNRTAVMIKDRWRTMKRLGMN 535 ZN282_HUMAN AEISLWTVVAAIQAVERKVDAQASQLLNLEGRTGTAEKKLADCEK TAVEFGNHMESKWAVLGTLLQEYGLLQRRLENLEN 536 RGS12_HUMAN LEKRTLFRLDLVPINRSVGLKAKPTKPVTEVLRPVVARYGLDLSG LLVRLSGEKEPLDLGAPISSLDGQRVVLEEKDPSR 537 ZN840_HUMAN PNCLSSSMQLPHGGGRHQELVRERDVAVVESPEEWDHLTPEQRNL YKDVMLDNCKYLASLGNWTYKAHVMSSLKQGKEPW 538 SPI2B_HUMAN DDYKEGDLRIMPESSESPPTEREPGGVVDGLIGKHVEYTKEDGSK RIGMVIHQVEAKPSVYFIKFDDDFHIYVYDLVKKS 539 PAX7_HUMAN SEPDLPLKRKQRRSRTTFTAEQLEELEKAFERTHYPDIYTREELA QRTKLTEARVQVWESNRRARWRKQAGANQLAAFNH 540 NKX62_HUMAN AGGVLDKDGKKKHSRPTFSGQQIFALEKTFEQTKYLAGPERARLA YSLGMTESQVKVWFQNRRTKWRKRHAVEMASAKKK 541 ASXL2_HUMAN DVMSFSVTVTTIPASQAMNPSSHGQTIPVQAFSEENSIEGTPSKC YCRLKAMIMCKGCGAFCHDDCIGPSKLCVSCLVVR 542 FOX01_HUMAN GGYSSVSSCNGYGRMGLLHQEKLPSDLDGMFIERLDCDMESIIRN DLMDGDTLDENEDNVLPNQSFPHSVKTTTHSWVSG 543 GATA3_HUMAN GGSPTGFGCKSRPKARSSTGRECVNCGATSTPLWRRDGTGHYLCN ACGLYHKMNGQNRPLIKPKRRLSAARRAGTSCANC 544 GATA1_HUMAN GQNRPLIRPKKRLIVSKRAGTQCTNCQTTTTTLWRRNASGDPVCN ACGLYYKLHQVNRPLTMRKDGIQTRNRKASGKGKK 545 ZMYM5_HUMAN PVALLRKQNFQPTAQQQLTKPAKITCANCKKPLQKGQTAYQRKGS AHLFCSTTCLSSFSHKRTQNTRSIICKKDASTKKA 546 ZN783_HUMAN TEITLWTVVAAIQALEKKVDSCLTRLLTLEGRTGTAEKKLADCEK TAVEFGNQLEGKWAVLGTLLQEYGLLQRRLENVEN 547 SPI2B_HUMAN KKQRGRPSSQPRRNIVGCRISHGWKEGDEPITQWKGTVLDQVPIN PSLYLVKYDGIDCVYGLELHRDERVLSLKILSDRV 548 LRP1_HUMAN WTCDLDDDCGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCD NDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHW 549 MIXL1_HUMAN PKGAAAPSASQRRKRTSFSAEQLQLLELVERRTRYPDIHLRERLA ALTLLPESRIQVWFQNRRAKSRRQSGKSFQPLARP 550 SGT1_HUMAN KIKYDWYQTESQVVITLMIKNVQKNDVNVEFSEKELSALVKLPSG EDYNLKLELLHPIIPEQSTEKVLSTKIEIKLKKPE 551 LMCDI_HUMAN DPSKEVEYVCELCKGAAPPDSPVVYSDRAGYNKQWHPTCFVCAKC SEPLVDLIYFWKDGAPWCGRHYCESLRPRCSGCDE 552 CEBPA_HUMAN GSGAGKAKKSVDKNSNEYRVRRERNNIAVRKSRDKAKQRNVETQQ KVLELTSDNDRLRKRVEQLSRELDTLRGIFRQLPE 553 GATA2_HUMAN GPASSFTPKQRSKARSCSEGRECVNCGATATPLWRRDGTGHYLCN ACGLYHKMNGQNRPLIKPKRRLSAARRAGTCCANC 554 SOX14_HUMAN KPSDHIKRPMNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWK LLSEAEKRPYIDEAKRLRAQHMKEHPDYKYRPRRK 555 WTIP_HUMAN LYSGFQQTADKCSVCGHLIMEMILQALGKSYHPGCFRCSVCNECL DGVPFTVDVENNIYCVRDYHTVFAPKCASCARPIL 556 PRP19_HUMAN HPSQDLVESASPDATIRIWSVPNASCVQVVRAHESAVTGLSLHAT GDYLLSSSDDQYWAFSDIQTGRVLTKVTDETSGCS 557 CBX6_HUMAN ELSAVGERVFAAESIIKRRIRKGRIEYLVKWKGWAIKYSTWEPEE NILDSRLIAAFEQKERERELYGPKKRGPKPKTELL 558 NKX11_HUMAN RTGSDSKSGKPRRARTAFTYEQLVALENKFKATRYLSVCERLNLA LSLSLTETQVKIWFQNRRTKWKKQNPGADTSAPTG 559 RBBP4_HUMAN VWDLSKIGEEQSPEDAEDGPPELLFIHGGHTAKISDESWNPNEPW VICSVSEDNIMQVWQMAENIYNDEDPEGSVDPEGQ 560 DMRT2_HUMAN ERCTPAGGGAEPRKLSRTPKCARCRNHGVVSCLKGHKRFCRWRDC QCANCLLVVERQRVMAAQVALRRQQATEDKKGLSG 561 SMCA2_HUMAN SQPGALIPGDPQAMSQPNRGPSPFSPVQLHQLRAQILAYKMLARG QPLPETLQLAVQGKRTLPGLQQQQQQQQQQQQQQQ 562 ZNF10 MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLE NYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETA FEIKSSVSSRSIFKDKQSCDIKMEGMARNDLWYLSLEEVWKCRDQ LDKYQENPERHLRQVAFTQKKVLTQERVSESGKYGGNCLLPAQLV LREYFHKRDSHTKSLKHDLVLNGHQDSCASNSNECGQTFCQNIHL IQFARTHTGDKSYKCPDNDNSLTHGSSLGISKGIHREKPYECKEC GKFFSWRSNLTRHQLIHTGEKPYECKECGKSFSRSSHLIGHQKTH TGEEPYECKECGKSFSWFSHLVTHQRTHTGDKLYTCNQCGKSFVH SSRLIRHQRTHTGEKPYECPECGKSFRQSTHLILHQRTHVRVRPY ECNECGKSYSQRSHLVVHHRIHTGLKPFECKDCGKCFSRSSHLYS HQRTHTGEKPYECHDCGKSFSQSSALIVHQRIHTGEKPYECCQCG KAFIRKNDLIKHQRIHVGEETYKCNQCGIIFSQNSPFIVHQIAHT GEQFLTCNQCGTALVNTSNLIGYQTNHIRENAY 563 EED_HUMAN MSEREVSTAPAGTDMPAAKKQKLSSDENSNPDLSGDENDDAVSIE SGTNTERPDTPTNTPNAPGRKSWGKGKWKSKKCKYSFKCVNSLKE DHNQPLFGVQFNWHSKEGDPLVFATVGSNRVTLYECHSQGEIRLL QSYVDADADENFYTCAWTYDSNTSHPLLAVAGSRGIIRIINPITM QCIKHYVGHGNAINELKFHPRDPNLLLSVSKDHALRLWNIQTDTL VAIFGGVEGHRDEVLSADYDLLGEKIMSCGMDHSLKLWRINSKRM MNAIKESYDYNPNKTNRPFISQKIHFPDESTRDIHRNYVDCVRWL GDLILSKSCENAIVCWKPGKMEDDIDKIKPSESNVTILGREDYSQ CDIWYMRFSMDFWQKMLALGNQVGKLYVWDLEVEDPHKAKCTTLT HHKCGAAIRQTSFSRDSSILIAVCDDASIWRWDRLR 564 RCORI_HUMAN MPAMVEKGPEVSGKRRGRNNAAASASAAAASAAASAACASPAATA ASGAAASSASAAAASAAAAPNNGQNKSLAAAAPNGNSSSNSWEEG SSGSSSDEEHGGGGMRVGPQYQAVVPDFDPAKLARRSQERDNLGM LVWSPNQNLSEAKLDEYIAIAKEKHGYNMEQALGMLFWHKHNIEK SLADLPNFTPFPDEWTVEDKVLFEQAFSFHGKTFHRIQQMLPDKS IASLVKFYYSWKKTRTKTSVMDRHARKQKREREESEDELEEANGN NPIDIEVDQNKESKKEVPPTETVPQVKKEKHSTQAKNRAKRKPPK GMFLSQEDVEAVSANATAATTVLRQLDMELVSVKRQIQNIKQTNS ALKEKLDGGIEPYRLPEVIQKCNARWTTEEQLLAVQAIRKYGRDF QAISDVIGNKSVVQVKNFFVNYRRRENIDEVLQEWEAEHGKEETN GPSNQKPVKSPDNSIKMPEEEDEAPVLDVRYASAS 565 KOX1/ZNF10 TGRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSL KRAB 1 GYQLTKPDVILRLEKGEEPLEINLWITKFVKD 566 KOX1/ZNF10 MYPYDVPDYASPKKKRKVGGGASMDAKSLTAWSRTLVTFKDVFVD KRAB 2 FTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLE KGEEPWLVEREIHQETHPDSETAFEIKSSV 567 KOX1/ZNF10 ALSPQHSAVTQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTR KRAB 3 EEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEKGE EPWLVEREIHQETHPDSETAFEIKSSV 568 KOX1/ZNF10 (aa RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY 11-72) QLTKPDVILRLEKGEEP 569 KOX1/ZNF10 (aa RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY 11-108) QLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSVSSR SIFKDKQS 570 KOX1/ZNF10 RTLVTFKDVAVDFTQEEWQQLDPAQKIVYRDVMLENYSNLVSVGY variant QLTKPDVILRLEQKGEEPWLVEEEIHQETHPDSETAFEIKSSVSS RSIFKDKQS 571 KOX1 KRAB-ZIM3 RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY chimera QLTKPDVILRLEKGEEPWLEEEEVLGSGRAEKNGDIGGQIWKPKD VKESL 572 ZIM3-KOX1 KRAB MNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVMLENYSNLV chimera SVGQGETTKPDVILRLEQGKEPWLVEREIHQETHPDSETAFEIKS SVSSRSIFKDKQS 573 human DNMT1 MPARTAPARVPTLAVPAISLPDDVRRRLKDLERDSLTEKECVKEK LNLLHEFLQTEIKNQLCDLETKLRKEELSEEGYLAKVKSLLNKDL SLENGAHAYNREVNGRLENGNQARSEARRVGMADANSPPKPLSKP RTPRRSKSDGEAKPEPSPSPRITRKSTRQTTITSHFAKGPAKRKP QEESERAKSDESIKEEDKDQDEKRRRVTSRERVARPLPAEEPERA KSGTRTEKEEERDEKEEKRLRSQTKEPTPKQKLKEEPDREARAGV QADEDEDGDEKDEKKHRSQPKDLAAKRRPEEKEPEKVNPQISDEK DEDEKEEKRRKTTPKEPTEKKMARAKTVMNSKTHPPKCIQCGQYL DDPLKYGQHPPDAVDEPQMLTNEKLSIFDANESGFESYEALPQHK LTCFSVYCKHGHLCPIDTGLIEKNIELFFSGSAKPIYDDDPSLEG GVNGKNLGPINEWWITGEDGGEKALIGFSTSFAEYILMDPSPEYA PIFGLMQEKIYISKIVVEFLQSNSDSTYEDLINKIETTVPPSGLN LNRFTEDSLLRHAQFVVEQVESYDEAGDSDEQPIFLTPCMRDLIK LAGVTLGQRRAQARRQTIRHSTREKDRGPTKATTTKLVYQIFDTE FAEQIEKDDREDKENAFKRRRCGVCEVCQQPECGKCKACKDMVKF GGSGRSKQACQERRCPNMAMKEADDDEEVDDNIPEMPSPKKMHQG KKKKQNKNRISWVGEAVKTDGKKSYYKKVCIDAETLEVGDCVSVI PDDSSKPLYLARVTALWEDSSNGQMFHAHWFCAGTDTVLGATSDP LELFLVDECEDMQLSYIHSKVKVIYKAPSENWAMEGGMDPESLLE GDDGKTYFYQLWYDQDYARFESPPKTQPTEDNKFKFCVSCARLAE MRQKEIPRVLEQLEDLDSRVLYYSATKNGILYRVGDGVYLPPEAF TFNIKLSSPVKRPRKEPVDEDLYPEHYRKYSDYIKGSNLDAPEPY RIGRIKEIFCPKKSNGRPNETDIKIRVNKFYRPENTHKSTPASYH ADINLLYWSDEEAVVDFKAVQGRCTVEYGEDLPECVQVYSMGGPN RFYFLEAYNAKSKSFEDPPNHARSPGNKGKGKGKGKGKPKSQACE PSEPEIEIKLPKLRTLDVFSGCGGLSEGFHQAGISDTLWAIEMWD PAAQAFRLNNPGSTVFTEDCNILLKLVMAGETTNSRGQRLPQKGD VEMLCGGPPCQGFSGMNRENSRTYSKFKNSLVVSFLSYCDYYRPR FFLLENVRNFVSFKRSMVLKLTLRCLVRMGYQCTFGVLQAGQYGV AQTRRRAIILAAAPGEKLPLFPEPLHVFAPRACQLSVVVDDKKFV SNITRLSSGPFRTITVRDTMSDLPEVRNGASALEISYNGEPQSWF QRQLRGAQYQPILRDHICKDMSALVAARMRHIPLAPGSDWRDLPN IEVRLSDGTMARKLRYTHHDRKNGRSSSGALRGVCSCVEAGKACD PAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNPE PMGKQGRVLHPEQHRVVSVRECARSQGFPDTYRLEGNILDKHRQV GNAVPPPLAKAIGLEIKLCMLAKARESASAKIKEEEAAKD 574 human DNMT3A MPAMPSSGPGDTSSSAAEREEDRKDGEEQEEPRGKEERQEPSTTA RKVGRPGRKRKHPPVESGDTPKDPAVISKSPSMAQDSGASELLPN GDLEKRSEPQPEEGSPAGGQKGGAPAEGEGAAETLPEASRAVENG CCTPKEGRGAPAEAGKEQKETNIESMKMEGSRGRLRGGLGWESSL RQRPMPRLTFQAGDPYYISKRKRDEWLARWKREAEKKAKVIAGMN AVEENQGPGESQKVEEASPPAVQQPTDPASPTVATTPEPVGSDAG DKNATKAGDDEPEYEDGRGFGIGELVWGKLRGESWWPGRIVSWWM TGRSRAAEGTRWVMWFGDGKFSVVCVEKLMPLSSFCSAFHQATYN KQPMYRKAIYEVLQVASSRAGKLFPVCHDSDESDTAKAVEVQNKP MIEWALGGFQPSGPKGLEPPEEEKNPYKEVYTDMWVEPEAAAYAP PPPAKKPRKSTAEKPKVKEIIDERTRERLVYEVRQKCRNIEDICI SCGSLNVTLEHPLFVGGMCQNCKNCFLECAYQYDDDGYQSYCTIC CGGREVLMCGNNNCCRCFCVECVDLLVGPGAAQAAIKEDPWNCYM CGHKGTYGLLRRREDWPSRLQMFFANNHDQEFDPPKVYPPVPAEK RKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMV RHQGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPAR KGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSD KRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVN DKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEK EDILWCTEMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRH LFAPLKEYFACV 575 human DNMT3A NHDQEFDPPKVYPPVPAEKRKPIRVLSLEDGIATGLLVLKDLGIQ catalytic domain VDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPF DLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEG DDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRA RYFWGNLPGMNRPLASTVNDKLELQECLEHGRIAKFSKVRTITTR SNSIKQGKDQHFPVEMNEKEDILWCTEMERVFGFPVHYTDVSNMS RLARQRLLGRSWSVPVIRHLFAPLKEYFACV 576 human DNMT3B MKGDTRHLNGEEDAGGREDSILVNGACSDQSSDSPPILEAIRTPE IRGRRSSSRLSKREVSSLLSYTQDLTGDGDGEDGDGSDTPVMPKL FRETRTRSESPAVRTRNNNSVSSRERHRPSPRSTRGRQGRNHVDE SPVEFPATRSLRRRATASAGTPWPSPPSSYLTIDLTDDTEDTHGT PQSSSTPYARLAQDSQQGGMESPQVEADSGDGDSSEYQDGKEFGI GDLVWGKIKGFSWWPAMVVSWKATSKRQAMSGMRWVQWEGDGKES EVSADKLVALGLFSQHENLATENKLVSYRKAMYHALEKARVRAGK TFPSSPGDSLEDQLKPMLEWAHGGFKPTGIEGLKPNNTQPVVNKS KVRRAGSRKLESRKYENKTRRRTADDSATSDYCPAPKRLKTNCYN NGKDRGDEDQSREQMASDVANNKSSLEDGCLSCGRKNPVSFHPLF EGGLCQTCRDRFLELFYMYDDDGYQSYCTVCCEGRELLLCSNTSC CRCFCVECLEVLVGTGTAAEAKLQEPWSCYMCLPQRCHGVLRRRK DWNVRLQAFFTSDTGLEYEAPKLYPAIPAARRRPIRVLSLEDGIA TGYLVLKELGIKVGKYVASEVCEESIAVGTVKHEGNIKYVNDVRN ITKKNIEEWGPFDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEF YHLLNYSRPKEGDDRPFFWMFENVVAMKVGDKRDISRFLECNPVM IDAIKVSAAHRARYFWGNLPGMNRPVIASKNDKLELQDCLEYNRI AKLKKVQTITTKSNSIKQGKNQLFPVVMNGKEDVLWCTELERIFG FPVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACE 577 mouse DNMT3C MRGGSRHLSNEEDVSGCEDCIIISGTCSDQSSDPKTVPLTQVLEA VCTVENRGCRTSSQPSKRKASSLISYVQDLTGDGDEDRDGEVGGS SGSGTPVMPQLFCETRIPSKTPAPLSWQANTSASTPWLSPASPYP IIDLTDEDVIPQSISTPSVDWSQDSHQEGMDTTQVDAESRDGGNI EYQVSADKLLLSQSCILAAFYKLVPYRESIYRTLEKARVRAGKAC PSSPGESLEDQLKPMLEWAHGGFKPTGIEGLKPNKKQPENKSRRR TTNDPAASESSPPKRLKTNSYGGKDRGEDEESREQMASDVTNNKG NLEDHCLSCGRKDPVSFHPLFEGGLCQSCRDRELELFYMYDEDGY QSYCTVCCEGRELLLCSNTSCCRCFCVECLEVLVGAGTAEDVKLQ EPWSCYMCLPQRCHGVLRRRKDWNMRLQDFFTTDPDLEEFEPPKL YPAIPAAKRRPIRVLSLFDGIATGYLVLKELGIKVEKYIASEVCA ESIAVGTVKHEGQIKYVDDIRNITKEHIDEWGPFDLVIGGSPCND LSCVNPVRKGLFEGTGRLFFEFYRLLNYSCPEEEDDRPFFWMFEN VVAMEVGDKRDISRFLECNPVMIDAIKVSAAHRARYFWGNLPGMN RPVMASKNDKLELQDCLEFSRTAKLKKVQTITTKSNSIRQGKNQL FPVVMNGKDDVLWCTELERIFGFPEHYTDVSNMGRGARQKLLGRS WSVPVIRHLFAPLKDHFACE 578 human DNMT3L MAAIPALDPEAEPSMDVILVGSSELSSSVSPGTGRDLIAYEVKAN QRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKELDALFLYDDD GYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVH AMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFE TVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVT DTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYA RPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGG SLQNAVRVWSNIPAIRSSRHWALVSEEELSLLAQNKQSSKLAAKW PTKLVKNCFLPLREYFKYFSTELTSSL 579 human DNMT3L NPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQL catalytic domain KHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQF HRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVT IPDVHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQSS KLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL 580 mouse DNMT3L MGSRETPSSCSKTLETLDLETSDSSSPDADSPLEEQWLKSSPALK EDSVDVVLEDCKEPLSPSSPPTGREMIRYEVKVNRRSIEDICLCC GTLQVYTRHPLFEGGLCAPCKDKFLESLFLYDDDGHQSYCTICCS GGTLFICESPDCTRCYCFECVDILVGPGTSERINAMACWVCFLCL PFSRSGLLQRRKRWRHQLKAFHDQEGAGPMEIYKTVSAWKRQPVR VLSLERNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEK WGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRP FFWIFMDNLLLTEDDQETTTRFLQTEAVTLQDVRGRDYQNAMRVW SNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLDAPKVDLLVKNCLL PLREYFKYFSQNSLPL 581 mouse DNMT3L GPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGG catalytic domain TLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMF QFHRILQYALPRQESQRPFFWIFMDNLLLTEDDQETTTRELQTEA VTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRS RSKLDAPKVDLLVKNCLLPLREYFKYFSQNSLPL 582 Ailuropoda MALSPTGTLSVETLDRSDPDPLDEGPWQATCEILLEPDAEHSTDV melanoleuca ILVGSSELSAPASPGPRRDLLAYEVKVNQRDIEDVCICCGSLRVH DNMT3L TQHPLFEGGMCAPCKDKELDCLFLYDDDGYQSYCSICCAGETLLI CENPDCTRPSLMMKLRLFRECACLIFPSEGMLLQTVWFWKMTVVW QPGLRHLPQENPLETYKTVPVWKREPVRVLSLEGDIRRELMSLGF LESGSAPGRLKHLDDVTDVVRKDVEGWGPFDLVYGSTPPIGHACD HPPVWYLLQFHRILQYARPRPGSQQPFFWMFVDNLVLSQDDQTAA TRFLEADPVTIQDVCGRAVRNTVHVWSNIPAVRSRHSALALCEEL SLLAQDRQRTKPPAQGPAQLVKNCELPLREYFKYFSTELTSSL 583 Ailuropoda NPLETYKTVPVWKREPVRVLSLEGDIRRELMSLGFLESGSAPGRL melanoleuca KHLDDVTDVVRKDVEGWGPFDLVYGSTPPIGHACDHPPVWYLLQF DNMT3L catalytic HRILQYARPRPGSQQPFFWMFVDNLVLSQDDQTAATRFLEADPVT domain IQDVCGRAVRNTVHVWSNIPAVRSRHSALALCEELSLLAQDRQRT KPPAQGPAQLVKNCFLPLREYFKYFSTELTSSL 584 Carlito syrichta MALSCRRTLPLESLHSSNSDLASQLDKEQWRPPCETHGIPVAAAP DNMT3L VLDLEAECSLDVILVGSSELSTSSSPRLGRDHIAYEVKVNQRNIE DICLCCGSFLVHTQHPLFEGGMCAPCKDKELDTLFLYDEDGYQSY CSICCSGETLLICENPDCTRCYCFECLDTLVSPGTSEKVHAMSNW VCFLCLPFTRSGLLQRRRKWRGQLKAFYDRESESSLEMYKTVPVW KREPVRVLSLFGDIKKELMSLGFVETGSDPGRLRHLDDTTNIVRR NVEEWGPFHLLYGATPPLGHTCDRPPGWYLFQFHRLLQYARPQPG SPQPFFWMFVDNVMLTREDRAIASRFLETEPVTIPDIHGRALQNA VCVWSNIPAVRSKHSALVSEEELSLLAQDRQRAKLPTQGPTKLVK NCFLPLREYFKYFSTELTSFL 585 Carlito syrichta SSLEMYKTVPVWKREPVRVLSLFGDIKKELMSLGFVETGSDPGRL DNMT3L catalytic RHLDDTTNIVRRNVEEWGPFHLLYGATPPLGHTCDRPPGWYLFQF domain HRLLQYARPQPGSPQPFFWMFVDNVMLTREDRAIASRFLETEPVT IPDIHGRALQNAVCVWSNIPAVRSKHSALVSEEELSLLAQDRQRA KLPTQGPTKLVKNCELPLREYFKYFSTELTSEL 586 Meriones MGSQETPSTRAKTPGTWNLESTDSSSPESLGHLEEQWANSSPDLK unguiculatus DEHSKDVEPEDSKELISSASPPSGREIIRYEISVNQRNIEDICLC DNMT3L CGTLQVYKQHPLFEGGICAPCKDKFLETFFLYDEDGHQSYCSICC SGGTLFICESPDCTRCYCFECVDILVGPGTSERINAMPCWVCFLC LPFTRSGLLQRRRKWRHQLKAFFDEGGASPLEMYKTVSAWKRKPM RVLSLFKNIDKELKNLGFLESGSGSEEERLKYLEDVTNVVRRDVE KWGPFDLVYGSTRPRGSSCDHCPAWYMFQFHRILQYARPPSGSEQ PFFWVFVDNLLMTEDDQITADRFLQMKAVTLQDVRGRVLQNAVRV WSNIPGVKSKHMALTEKEEQSLEAQAGTRTKLSAQKVDPLVKNCL LPLREYFKFFSQNSLPLDK 587 Meriones SPLEMYKTVSAWKRKPMRVLSLFKNIDKELKNLGFLESGSGSEEE unguiculatus RLKYLEDVTNVVRRDVEKWGPFDLVYGSTRPRGSSCDHCPAWYME DNMT3L catalytic QFHRILQYARPPSGSEQPFFWVFVDNLLMTEDDQITADRELQMKA domain VTLQDVRGRVLQNAVRVWSNIPGVKSKHMALTEKEEQSLEAQAGT RTKLSAQKVDPLVKNCLLPLREYFKFFSQNSLPLDK 588 Ochotona princeps MALPSPETLDSLDRVPASHPDEQHWTVCDNSDPILEVEAEGSMDV DNMT3L ILVDDSPAPSGRDRIELEVKVNQRSIEDLCLCCGSSQVHRQHPLE QGGLCAPCKDKFLEALFLYDEDGYQSYCSICGLGDTLLVCESPDC TRGYCFACVDGLVGAGSSGHMHTVSPWVCFLCVPGSRHGLLQRRR RWRTQLKVFHEQEAAQPLEIYETVPACRRKPLRVLSLFEHIEKEL ASLGFLETGSSPGRIRHLDDVTDVVRRDVEQWGPFDLVYGSTPPL GHASPRSPGWYLFQFHRMLQYTQPTASTQRPFFWMFVDNLLLTRD DLVTATRFLEVEPATLQDVRGRVLQGAMRVWSNIPAVNSRHTELA PEAETALLAQSCRRAKASGEGLARLLKSCELPLREYFKYFPQSPL PLRK 589 Ochotona princeps QPLEIYETVPACRRKPLRVLSLFEHIEKELASLGFLETGSSPGRI DNMT3L catalytic RHLDDVTDVVRRDVEQWGPFDLVYGSTPPLGHASPRSPGWYLFQF domain HRMLQYTQPTASTQRPFFWMFVDNLLLTRDDLVTATRELEVEPAT LQDVRGRVLQGAMRVWSNIPAVNSRHTELAPEAETALLAQSCRRA KASGEGLARLLKSCELPLREYFKYFPQSPLPLRK 590 Neosciurus MGGPRPAAVEESPHEIYKTVPAWKREPMRVLSLFGDIGKELTSLG carolinensis FLETGSEAGRLKHLEDVTDTVRRDVEEWGPFDLVYGSTPALGHSC DNMT3L DRSPGWYLFQFHRLLQYARPRLGSPKPFFWMFVDNLLLTKDDQAI ASRFLEMEPVTLQDVHGRVLQNAVRVWTNVPAVKSRHSALASEEE LLLVQDGQRGRLPAQGPAALVKHCFLPLREYFKYFSQNTLPLYK 591 Neosciurus SPHEIYKTVPAWKREPMRVLSLFGDIGKELTSLGFLETGSEAGRL carolinensis KHLEDVTDTVRRDVEEWGPFDLVYGSTPALGHSCDRSPGWYLFQF DNMT3L catalytic HRLLQYARPRLGSPKPFFWMFVDNLLLTKDDQAIASRFLEMEPVT domain LQDVHGRVLQNAVRVWTNVPAVKSRHSALASEEELLLVQDGQRGR LPAQGPAALVKHCELPLREYFKYFSQNTLPLYK 592 Bison bison MARSSPGTLNLEIMDGSDPDPALPPDREQWPPPCEILLDPEPEHS DNMT3L LDIILVGSSELSSPPSPGPRRDFIAYEVKVNQRDIEDVCICCGSL QLHTQHPLFEGGMCAPCKDKFLECLFLYDDDGYQSYCSICCAGET LLICENPDCTRCYCFECVDTLVGPGTSGKVHAMSNWVCFLCLPFP RSGLLQRRRKWRTWLKAFYDREAESPLVMYKTVPVWKREPIRVLS LFGDIKKELTSLGFLEDGSKPGRLKHLDDVTNIVRRDIDEWGPED LTYGSTPTLGHTCDHPPGWYVYQFHRILQYARPLPGSPQPFFWME VDNLVLTEEDLDVATRFLETDPVTIQDVRGRTVQNAVHVWSNIPA VKSRHSALVSQEELSLLAQDRQRVKSPVQGPATLVKNCFLPLREY FKYFSTELTSSL 593 Bison bison SPLVMYKTVPVWKREPIRVLSLFGDIKKELTSLGFLEDGSKPGRL DNMT3L catalytic KHLDDVTNIVRRDIDEWGPFDLTYGSTPTLGHTCDHPPGWYVYQF domain HRILQYARPLPGSPQPFFWMFVDNLVLTEEDLDVATRFLETDPVT IQDVRGRTVQNAVHVWSNIPAVKSRHSALVSQEELSLLAQDRQRV KSPVQGPATLVKNCELPLREYFKYFSTELTSSL 594 Equus przewalskii MALSSPGTLSLETLDSWDPDVAGQLDEERWQPSSEIVGRPMAAAP DNMT3L VLDLEEEPSMDIILVDSSELSSPPSPGPSRDMCICCGSFQVHTQH PLFEGGMCAACKDKFLSCLFLYDDDGNQSYCSICCSGETLLICEN PDCTRCYCFECVDTLVSPRTSEKVQAMSNWVCFLCLPFPRSGLLQ RRRKWRGWLKAFYDQEAVRSRSAWGRRMRSGPHLVGFLWLLVAKC PSALESPLEMYKTVPVWKREPVRVLSLFGDIKKELTTLGFLENGS DPGRLKHLDDVTNTVRRDVEEWGPFDLVYGSTPPLGHACDHPPGW YLFQFHRVLQYARPRPGSPQAFFWMFVDNLVLTEDDRAVATRELE TDPVTIQDVCGRAVRNAVHVWSNIPAVKSRHSALESQEESFLRAQ DRQRAKPPARGPAKLVKNCFLPLREYFKYESTEFTSSL 595 Equus przewalskii SPLEMYKTVPVWKREPVRVLSLFGDIKKELTTLGFLENGSDPGRL DNMT3L catalytic KHLDDVTNTVRRDVEEWGPFDLVYGSTPPLGHACDHPPGWYLFQF domain HRVLQYARPRPGSPQAFFWMFVDNLVLTEDDRAVATRFLETDPVT IQDVCGRAVRNAVHVWSNIPAVKSRHSALESQEESFLRAQDRQRA KPPARGPAKLVKNCFLPLREYFKYESTEFTSSL 596 Mus caroli MGSRETPSSFSKTLETLDLETSDSSSPDADSPLEEQWLKSSPALK DNMT3L EDNVDMVLEDCKEPLSPSSPPTGREMIRYEVKVNRRSIEDICLCC GTLQVYTQHPLFEGGICAPCKDKFLESLFLYDDDGHQSYCTICCS GGTLFICESPDCTRCYCFECVDILVGPGTSERINAMACWVCFLCL PFSRSGLLQRRKRWRHQLKAFHDQEGAGPMEIYKTVSTWKRQPVR VLSLFGNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVRRDVEK WGPFDLVYGSTQPLGSSCDRCPGWYMFQFHRILQYALPRQESQRP FFWIFMDNLLMTEDDQETTARFLQTEAVTLQDVRGRDYQNVMRVW SNIPGLKSKHVPLTPKEEEYLQAQVRTRSKLDAQKVDLLVKNCLL PLREYFKYFS 597 Mus caroli GPMEIYKTVSTWKRQPVRVLSLFGNIDKVLKSLGFLESGSGSGGG DNMT3L catalytic TLKYVEDVTNVVRRDVEKWGPFDLVYGSTQPLGSSCDRCPGWYMF domain QFHRILQYALPRQESQRPFFWIFMDNLLMTEDDQETTARFLQTEA VTLQDVRGRDYQNVMRVWSNIPGLKSKHVPLTPKEEEYLQAQVRT RSKLDAQKVDLLVKNCLLPLREYFKYFS 598 Pan troglodytes MAAIPALDPEAEPSMDVILVGSSELSSSISPRTGRDLIAYEVKAN DNMT3L QRNIEDICICCGSLQVHTQHPLFEGGICAPCKDKSLDALFLYDDD GYQSYCSICCSGETLLICGNPDCTRCYCFECVDSLVGPGTSGKVH AMSNWVCYLCLPSSRSGLLQRRRKWRSQLKAFYDRESENPLEMFE TVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVT DTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYA RPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPDVHGG SLQNAVRVWSNIPAIRSSRHWALVSEEELSLLAQNKQSSKLAAKW PTKLVKNCFLPLREYFKYFSTELTSSL 599 Pan troglodytes NPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQL DNMT3L catalytic KHVVDVTDTVRKDVEEWGPFDLVYGATPPLGHTCDRPPSWYLFQF domain HRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVT IPDVHGGSLQNAVRVWSNIPAIRSSRHWALVSEEELSLLAQNKQS SKLAAKWPTKLVKNCFLPLREYFKYFSTELTSSL 600 human TRDMT1 MEPLRVLELYSGVGGMHHALRESCIPAQVVAAIDVNTVANEVYKY (DNMT2) NFPHTQLLAKTIEGITLEEFDRLSEDMILMSPPCQPFTRIGRQGD MTDSRTNSFLHILDILPRLQKLPKYILLENVKGFEVSSTRDLLIQ TIENCGFQYQEFLLSPTSLGIPNSRLRYFLIAKLQSEPLPFQAPG QVLMEFPKIESVHPQKYAMDVENKIQEKNVEPNISEDGSIQCSGK DAILFKLETAEEIHRKNQQDSDLSVKMLKDFLEDDTDVNQYLLPP KSLLRYALLLDIVQPTCRRSVCFTKGYGSYIEGTGSVLQTAEDVQ VENIYKSLTNLSQEEQITKLLILKLRYFTPKEIANLLGFPPEFGF PEKITVKQRYRLLGNSLNVHVVAKLIKILYE 601 M. bacterium MAEWYIPAIVSYQAIHNGFTLNKINHKIELQTMIDYLESKTLSMN methyltransferase SKEPVKRGFWYKKHLDEIRIVYTAVKMSEQEGNIEDVRTLFERGL SDIDLLTYSFPCQDLSQQGKQKGMGRDSQTRSGLLWEIEKALDTS KKEDLPKYLLMENVVALTHKVNAEELDEWMMKLESLGYKNDLRIL NAGDFGSSQARRRTFMISTLNEKVELPVGNKKPKSMNKILNDEPT RKDFLPALDKEDLTEYKWTKSNINKAKLINYSTENSEAYVYDSNE TGPTLTASGANSRIKFEYNGKIRKIGAEEAYAYMGFKKSDYIKVN KLNYLNETKMIYTCGNSISVEVLRSIMTNINNNFKENK 602 M. marinum MLFLIGTFKYVLIYITKVIRIFEAFAGIGAQRKALRNIKSNYEVS methyltransferase GMAEWYIPAIVSYQAIHNGFTLSRVDKKTKLTEMIKYLESKTLSM DSKEPVRTGYWFKKHKDMVRIVYSAVKLSEAEGNIFDVRTLHERK LEDIDLLTYSFPCQDLSQQGKQRGMKKDSGTRSGLLWEIEKALEA TPKDKLPKYLLMENVVALTHKTNKKDLDNWKRKLRSLGYYNDINV LNAGDEGSSQARRRAFMISTLDSKVTLPLGDKKPQAISKILNKET RSQDEMPALDEYEKTDFKRTLSNIKKCKLIDYTSENSEAYVYDPK YTGPTLTASGANSRIKFTHQGKMRKINAEEAYRYMGESTNDYKKV NNLNFLSETKMIYTCGNSISVEVLEEIMLKIIREDNNG 603 S. chinense MKKIRLFEAFAGIGSQRRALKSVVGNNFEIAGLAEWYVPAIVMYQ methyltransferase IINNDESKKNVLDNVPRDEVIDYLNSKCLSWDSKKPVSKNEWNRK SQDILNVIYSAVKKSEEEGNIFDVRTLHERTLESIDILTYSFPCQ DLSQQGIQKGMKKNSGTRSGLLWEIEKAIDNTPKNNLPKILLMEN VPALLNKTNELELKEWLIKLENMGYKNSIGILNAADFGSPQARRR VFMISSRNKKIELPVGKSKPGKLNDILEKNVEDKFIMTNLEKYDE SEFSLTKSNIKKCSLINYTKFNSEAYVYDPDFTGPTLTASGANSR IKIYDKGFIRRMSPLESFRYMGEDDEDYKKIDEFEFLTDTQKIFV CGNSISIEVLKAIFERIDSNE 604 M. penetrans M MNSNKDKIKVIKVFEAFAGIGSQFKALKNIARSKNWEIQHSGMVE MpeI WFVDAIVSYVAIHSKNENPKIEQLDKDILSISNDSKMPISEYGIK KINNTIKASYLNYAKKHENNLFDIKKVNKDNFPKNIDIFTYSFPC QDLSVQGLQKGIDKELNTRSGLLWEIERILEEIKNSFSKEEMPKY LLMENVKNLLSHKNKKNYNTWLKQLEKFGYKSKTYLLNSKNEDNC QNRERVFCLSIRDDYLEKTGFKFKELEKVKNPPKKIKDILVDSSN YKYLNLNKYETTTFRETKSNIISRSLKNYTTENSENYVYNINGIG PTLTASGANSRIKIETQQGVRYLTPLECFKYMQFDVNDFKKVQST NLISENKMIYIAGNSIPVKILEAIENTLEFVNNEE 605 S. monobiae M SssI MSKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYV PAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSWNSKNPVS NGYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTLKNIDLL TYSFPCQDLSQQGIQKGMKRGSGTRSGLLWEIERALDSTEKNDLP KYLLMENVGALLHKKNEEELNQWKQKLESLGYQNSIEVLNAADEG SSQARRRVEMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILN NLLKYNLTEFKKTKSNINKASLIGYSKENSEGYVYDPEFTGPTLT ASGANSRIKIKDGSNIRKMNSDETFLYIGEDSQDGKRVNEIEFLT ENQKIFVCGNSISVEVLEAIIDKIGG 606 H. parainfluenzae M MKDVLDDNLLEEPAAQYSLFEPESNPNLREKFTFIDLFAGIGGER HpaII IAMQNLGGKCIFSSEWDEQAQKTYEANFGDLPYGDITLEETKAFI PEKFDILCAGFPCQAFSIAGKRGGFEDTRGTLFFDVAEIIRRHQP KAFFLENVKGLKNHDKGRTLKTILNVLREDLGYFVPEPAIVNAKN FGVPQNRERIYIVGFHKSTGVNSFSYPEPLDKIVTFADIREEKTV PTKYYLSTQYIDTLRKHKERHESKGNGFGYEIIPDDGIANAIVVG GMGRERNLVIDHRITDFTPTTNIKGEVNREGIRKMTPREWARLQG FPDSYVIPVSDASAYKQFGNSVAVPAIQATGKKILEKLGNLYD 607 A. luteus M AluI MSKANAKYSFVDLFAGIGGFHAALAATGGVCEYAVEIDREAAAVY ERNWNKPALGDITDDANDEGVTLRGYDGPIDVLTGGFPCQPFSKS GAQHGMAETRGTLFWNIARIIEEREPTVLILENVRNLVGPRHRHE WLTIIETLRFFGYEVSGAPAIFSPHLLPAWMGGTPQVRERVFITA TLVPERMRDERIPRTETGEIDAEAIGPKPVATMNDREPIKKGGTE LFHPGDRKSGWNLLTSGIIREGDPEPSNVDLRLTETETLWIDAWD DLESTIRRATGRPLEGFPYWADSWTDFRELSRLVVIRGFQAPERE VVGDRKRYVARTDMPEGFVPASVTRPAIDETLPAWKQSHLRRNYD FFERHFAEVVAWAYRWGVYTDLFPASRRKLEWQAQDAPRLWDTVM HFRPSGIRAKRPTYLPALVAITQTSIVGPLERRLSPRETARLQGL PEWFDFGEQRAAATYKQMGNGVNVGVVRHILREHVRRDRALLKLT PAGQRIINAVLADEPDATVGALGAAE 608 H. aegyptius M MNLISLFSGAGGLDLGFQKAGFRIICANEYDKSIWKTYESNHSAK HaeIII LIKGDISKISSDEFPKCDGIIGGPPCQSWSEGGSLRGIDDPRGKL FYEYIRILKQKKPIFFLAENVKGMMAQRHNKAVQEFIQEFDNAGY DVHIILLNANDYGVAQDRKRVFYIGFRKELNINYLPPIPHLIKPT FKDVIWDLKDNPIPALDKNKTNGNKCIYPNHEYFIGSYSTIFMSR NRVRQWNEPAFTVQASGRQCQLHPQAPVMLKVSKNLNKFVEGKEH LYRRLTVRECARVQGFPDDFIFHYESLNDGYKMIGNAVPVNLAYE IAKTIKSALEICKGN 609 H. haemolyticus M MIEIKDKQLTGLRFIDLFAGLGGFRLALESCGAECVYSNEWDKYA HhaI QEVYEMNFGEKPEGDITQVNEKTIPDHDILCAGEPCQAFSISGKQ KGFEDSRGTLFFDIARIVREKKPKVVFMENVKNFASHDNGNTLEV VKNTMNELDYSFHAKVLNALDYGIPQKRERIYMICERNDLNIQNE QFPKPFELNTFVKDLLLPDSEVEHLVIDRKDLVMTNQEIEQTTPK TVRLGIVGKGGQGERIYSTRGIAITLSAYGGGIFAKTGGYLVNGK TRKLHPRECARVMGYPDSYKVHPSTSQAYKQFGNSVVINVLQYIA YNIGSSLNEKPY 610 Moraxella M MspI MKPEILKLIRSKLDLTQKQASEIIEVSDKTWQQWESGKTEMHPAY YSFLQEKLKDKINFEELSAQKTLQKKIFDKYNQNQITKNAEELAE ITHIEERKDAYSSDFKFIDLESGIGGIRQSFEVNGGKCVESSEID PFAKFTYYTNFGVVPFGDITKVEATTIPQHDILCAGEPCQPFSHI GKREGFEHPTQGTMFHEIVRIIETKKTPVLFLENVPGLINHDDGN TLKVIIETLEDMGYKVHHTVLDASHFGIPQKRKRFYLVAFLNQNI HFEFPKPPMISKDIGEVLESDVTGYSISEHLQKSYLFKKDDGKPS LIDKNTTGAVKTLVSTYHKIQRLTGTFVKDGETGIRLLTTNECKA IMGFPKDFVIPVSRTQMYRQMGNSVVVPVVTKIAEQISLALKTVN QQSPQENFELELV 611 Ascobolus Masc1 MSERRYEAGMTVALHEGSELKIQRVYIRQYHADNRREHMLVGPLF RRTKYLKALSKKVNEVAIVHESIHVPVQDVIGVRELIITNRPFPE CRKGDEHTGRLVCRWVYNLDERAKGREYKKQRYIRRITEAEADPE YRVEDRVLRRRWFQEGYIGDEISYKEHGNGDIVDIRSESPLQVLD GWGGDLVDLENGEETSIPGPCRSASSYGRLMKPPLAQAADSNTSR KYTFGDTFCGGGGVSLGARQAGLEVKWAFDMNPNAGANYRRNEPN TDFFLAEAEQFIQLSVGISQHVDILHLSPPCQTFSRAHTIAGKND ENNEASFFAVVNLIKAVRPRLFTVEETDGIMDRQSRQFIDTALMG ITELGYSFRICVLNAIEYGVCQNRKRLIIIGAAPGEELPPEPLPT HQDFFSKDPRRDLLPAVTLDDALSTITPESTDHHLNHVWQPAEWK TPYDAHRPFKNAIRAGGGEYDIYPDGRRKFTVRELACIQGFPDEY EFVGTLTDKRRIIGNAVPPPLSAAIMSTLRQWMTEKDFERME 612 Arabidopsis MET1 MVENGAKAAKRKKRPLPEIQEVEDVPRTRRPRRAAACTSFKEKSI RVCEKSATIEVKKQQIVEEEFLALRLTALETDVEDRPTRRLNDEV LFDSDGVPQPLEMLEIHDIFVSGAILPSDVCTDKEKEKGVRCTSF GRVEHWSISGYEDGSPVIWISTELADYDCRKPAASYRKVYDYFYE KARASVAVYKKLSKSSGGDPDIGLEELLAAVVRSMSSGSKYFSSG AAIIDFVISQGDFIYNQLAGLDETAKKHESSYVEIPVLVALREKS SKIDKPLQRERNPSNGVRIKEVSQVAESEALTSDQLVDGTDDDRR YAILLQDEENRKSMQQPRKNSSSGSASNMFYIKINEDEIANDYPL PSYYKTSEEETDELILYDASYEVQSEHLPHRMLHNWALYNSDLRE ISLELLPMKQCDDIDVNIFGSGVVTDDNGSWISLNDPDSGSQSHD PDGMCIFLSQIKEWMIEFGSDDIISISIRTDVAWYRLGKPSKLYA PWWKPVLKTARVGISILTFLRVESRVARLSFADVTKRLSGLQAND KAYISSDPLAVERYLVVHGQIILQLFAVYPDDNVKRCPFVVGLAS KLEDRHHTKWIIKKKKISLKELNLNPRAGMAPVASKRKAMQATTT RLVNRIWGEFYSNYSPEDPLQATAAENGEDEVEEEGGNGEEEVEE EGENGLTEDTVPEPVEVQKPHTPKKIRGSSGKREIKWDGESLGKT SAGEPLYQQALVGGEMVAVGGAVTLEVDDPDEMPAIYFVEYMFES TDHCKMLHGRFLQRGSMTVLGNAANERELFLTNECMTTQLKDIKG VASFEIRSRPWGHQYRKKNITADKLDWARALERKVKDLPTEYYCK SLYSPERGGFFSLPLSDIGRSSGFCTSCKIREDEEKRSTIKLNVS KTGFFINGIEYSVEDFVYVNPDSIGGLKEGSKTSFKSGRNIGLRA YVVCQLLEIVPKESRKADLGSFDVKVRRFYRPEDVSAEKAYASDI QELYFSQDTVVLPPGALEGKCEVRKKSDMPLSREYPISDHIFFCD LFFDTSKGSLKQLPANMKPKFSTIKDDTLLRKKKGKGVESEIESE IVKPVEPPKEIRLATLDIFAGCGGLSHGLKKAGVSDAKWAIEYEE PAGQAFKQNHPESTVFVDNCNVILRAIMEKGGDQDDCVSTTEANE LAAKLTEEQKSTLPLPGQVDFINGGPPCQGFSGMNRENQSSWSKV QCEMILAFLSFADYFRPRYELLENVRTFVSENKGQTFQLTLASLL EMGYQVRFGILEAGAYGVSQSRKRAFIWAAAPEEVLPEWPEPMHV FGVPKLKISLSQGLHYAAVRSTALGAPFRPITVRDTIGDLPSVEN GDSRTNKEYKEVAVSWFQKEIRGNTIALTDHICKAMNELNLIRCK LIPTRPGADWHDLPKRKVTLSDGRVEEMIPFCLPNTAERHNGWKG LYGRLDWQGNFPTSVTDPQPMGKVGMCFHPEQHRILTVRECARSQ GFPDSYEFAGNINHKHRQIGNAVPPPLAFALGRKLKEALHLKKSP QHQP 613 Ascobolus Masc2 MELTPELSGVSTDLGGGGSIFAHWRMKEESPAPTEILDDLNVLEW EKTTRDYSKEDLRIADQLESIEDEHQSLPFETADAEDGTPTEEEE EKELPMRTLDNFVLYDASDLELAALDLIGTELNIHAVGTVGPIYT EGEEDEQEDEDEDVSPPVRTGTQATSASVTQMTVELYIRNIVQYE FCFNDDGTVETWIQTTNAHYKLLQPAKCYTSLYRPVNDCLNVITA IITLAPESTTMSLKDLLKVMDDKAQAVSYEEVERMSEFIVQHLDQ WMETAPKKKSKLIEKSKVYIDLNNLAGIDMVSGVRPPPVRRVTGR SSAPKKRIVRNMNDAVLLHQNETTVTNWIHQLSAGMFGRALNVLG AETADVENLTCDPASAKFVVPQRRLHKRLKWETRGHIPVSEEEYK HIYQGKKYAKFFEAVRAVDESKLTIKLGDLVYVLDQDPKVTQTQF ATAGREGRKKGAEKEKIQVRFGRVLSIRQPDSNSKDAQNVFIHVQ WLVLGCDTILQEMASRRELFLTDSCDTVFADVIYGVAKLTPLGAK DIPTVEFHESMATMMGENEFFVRFKYNYQDGSFTDLKDVDAEQIG TLQPRVNTHRNPGYCSNCRIKYDNERTGDKWIYENDTEGEPRLER SSKGWCIYAQEFVYLQPVEKQPGTTERVGYISEINKSSVIVELLA RVDDDDKSGHISYSDPRHLYFTGTDIKVTEDKIIRKCFVFHDSGD QKAKAPLMYGTLQRDLYYYRYEKRKGKAELVPVREIRSIHEQTLN DWESRTQIERHGAVSGKKLKGLDIFAGCGGLTLGLDLSGAVDTKW DIEFAPSAANTLALNEPDAQVENQCANVLLSRAIQSEDEGSLDIE YDLQGRVLPDLPKKGEVDFIYGGPPCQGFSGVNRYKKGNDIKNSL VATFLSYVDHYKPRFVLLENVKGLITTKLGNSKNAEGKWEGGISN GVVKFIYRTLISMNYQCRIGLVQSGEYGVPQSRPRVIFLAARMGE RLPDLPEPMHAFEVLDSQYALPHIKRYHTTQNGVAPLPRITIGEA VSDLPKFQYANPGVWPRHDPYSSAKAQPSDKTIEKFSVSKATSFV GYLLQPYHSRPQSEFQRRLRTKLVPSDEPAEKTSLLTTKLVTAHV TRLFNKETTQRIVCVPMWPGADHRSLPKEMRPWCLVDPNSQAEKH RFWPGLFGRLGMEDFFSTALTDVQPCGKQGKVLHPTQRRVYTVRE LARAQGFPDWFAFTDGDADSGLGGVKKWHRNIGNAVPVPLGEQIG RCIGYSVWWKDDMIAQLREDGADEDEEMIDGNDQWVEELNTQMAA DMPGLPLLVTHLLNLCVYRRLYGPNAKEFLPARVYDKKLEGGRRR LVWAML 614 Neurospora Dim2 MDSPDRSHGGMFIDVPAETMGFQEDYLDMFASVLSQGLAKEGDYA HHQPLPAGKEECLEPIAVATTITPSPDDPQLQLQLELEQQFQTES GLNGVDPAPAPESEDEADLPDGFSDESPDDDFVVQRSKHITVDLP VSTLINPRSTFQRIDENDNLVPPPQSTPERVAVEDLLKAAKAAGK NKEDYIEFELHDENFYVNYAYHPQEMRPIQLVATKVLHDKYYEDG VLKYGNTKHYVTGMQVLELPVGNYGASLHSVKGQIWVRSKHNAKK EIYYLLKKPAFEYQRYYQPFLWIADLGKHVVDYCTRMVERKREVT LGCFKSDFIQWASKAHGKSKAFQNWRAQHPSDDERTSVAANIGYI WKEINGVAGAKRAAGDQLFRELMIVKPGQYFRQEVPPGPVVTEGD RTVAATIVTPYIKECFGHMILGKVLRLAGEDAEKEKEVKLAKRLK IENKNATKADTKDDMKNDTATESLPTPLRSLPVQVLEATPIESDI VSIVSSDLPPSENNPPPLINGSVKPKAKANPKPKPSTQPLHAAHV KYLSQELVNKIKVGDVISTPRDDSSNTDTKWKPTDTDDHRWFGLV QRVHTAKTKSSGRGLNSKSFDVIWFYRPEDTPCCAMKYKWRNELF LSNHCTCQEGHHARVKGNEVLAVHPVDWFGTPESNKGEFFVRQLY ESEQRRWITLQKDHLTCYHNQPPKPPTAPYKPGDTVLATLSPSDK FSDPYEVVEYFTQGEKETAFVRLRKLLRRRKVDRQDAPANELVYT EDLVDVRAERIVGKCIMRCFRPDERVPSPYDRGGTGNMFFITHRQ DHGRCVPLDTLPPTLRQGENPLGNLGKPKLRGMDLYCGGGNFGRG LEEGGVVEMRWANDIWDKAIHTYMANTPDPNKTNPFLGSVDDLLR LALEGKFSDNVPRPGEVDFIAAGSPCPGFSLLTQDKKVLNQVKNQ SLVASFASFVDFYRPKYGVLENVSGIVQTFVNRKQDVLSQLFCAL VGMGYQAQLILGDAWAHGAPQSRERVELYFAAPGLPLPDPPLPSH SHYRVKNRNIGFLCNGESYVQRSFIPTAFKFVSAGEGTADLPKIG DGKPDACVRFPDHRLASGITPYIRAQYACIPTHPYGMNFIKAWNN GNGVMSKSDRDLFPSEGKTRTSDASVGWKRLNPKTLFPTVTTTSN PSDARMGPGLHWDEDRPYTVQEMRRAQGYLDEEVLVGRTTDQWKL VGNSVSRHMALAIGLKFREAWLGTLYDESAVVATATATATTAAAV GVTVPVMEEPGIGTTESSRPSRSPVHTAVDLDDSKSERSRSTTPA TVLSTSSAAGDGSANAAGLEDDDNDDMEMMEVTRKRSSPAVDEEG MRPSKVQKVEVTVASPASRRSSRQASRNPTASPSSKASKATTHEA PAPEELESDAESYSETYDKEGEDGDYHSGHEDQYSEEDEEEEYAE PETMTVNGMTIVKL 615 Drosophila dDnmt2 MVFRVLELESGIGGMHYAFNYAQLDGQIVAALDVNTVANAVYAHN YGSNLVKTRNIQSLSVKEVTKLQANMLLMSPPCQPHTRQGLQRDT EDKRSDALTHLCGLIPECQELEYILMENVKGFESSQARNQFIESL ERSGFHWREFILTPTQENVPNTRYRYYCIARKGADFPFAGGKIWE EMPGAIAQNQGLSQIAEIVEENVSPDFLVPDDVLTKRVLVMDIIH PAQSRSMCFTKGYTHYTEGTGSAYTPLSEDESHRIFELVKEIDTS NQDASKSEKILQQRLDLLHQVRLRYFTPREVARLMSFPENFEFPP ETTNRQKYRLLGNSINVKVVGELIKLLTIK 616 S. pombe Pmt1 MLSTKRLRVLELYSGIGGMHYALNLANIPADIVCAIDINPQANEI YNLNHGKLAKHMDISTLTAKDFDAFDCKLWTMSPSCQPFTRIGNR KDILDPRSQAFLNILNVLPHVNNLPEYILIENVQGFEESKAAEEC RKVLRNCGYNLIEGILSPNQFNIPNSRSRWYGLARLNEKGEWSID DVFQFSEVAQKEGEVKRIRDYLEIERDWSSYMVLESVLNKWGHQF DIVKPDSSSCCCFTRGYTHLVQGAGSILQMSDHENTHEQFERNRM ALQLRYFTAREVARLMGFPESLEWSKSNVTEKCMYRLLGNSINVK VVSYLISLLLEPLNE 617 Arabidopsis DRM1 MVMSHIFLISQIQEVEHGDSDDVNWNTDDDELAIDNFQFSPSPVH ISATSPNSIQNRISDETVASFVEMGESTQMIARAIEETAGANMEP MMILETLFNYSASTEASSSKSKVINHFIAMGFPEEHVIKAMQEHG DEDVGEITNALLTYAEVDKLRESEDMNININDDDDDNLYSLSSDD EEDELNNSSNEDRILQALIKMGYLREDAAIAIERCGEDASMEEVV DFICAAQMARQFDEIYAEPDKKELMNNNKKRRTYTETPRKPNTDQ LISLPKEMIGFGVPNHPGLMMHRPVPIPDIARGPPFFYYENVAMT PKGVWAKISSHLYDIVPEFVDSKHFCAAARKRGYIHNLPIQNRFQ IQPPQHNTIQEAFPLTKRWWPSWDGRTKLNCLLTCIASSRLTEKI REALERYDGETPLDVQKWVMYECKKWNLVWVGKNKLAPLDADEME KLLGFPRDHTRGGGISTTDRYKSLGNSFQVDTVAYHLSVLKPLFP NGINVLSLFTGIGGGEVALHRLQIKMNVVVSVEISDANRNILRSF WEQTNQKGILREFKDVQKLDDNTIERLMDEYGGFDLVIGGSPCNN LAGGNRHHRVGLGGEHSSLFFDYCRILEAVRRKARHMRR 618 Arabadopsis DRM2 MVIWNNDDDDFLEIDNFQSSPRSSPIHAMQCRVENLAGVAVTTSS LSSPTETTDLVQMGFSDEVFATLFDMGFPVEMISRAIKETGPNVE TSVIIDTISKYSSDCEAGSSKSKAIDHFLAMGEDEEKVVKAIQEH GEDNMEAIANALLSCPEAKKLPAAVEEEDGIDWSSSDDDTNYTDM LNSDDEKDPNSNENGSKIRSLVKMGFSELEASLAVERCGENVDIA ELTDELCAAQMAREFSEFYTEHEEQKPRHNIKKRRFESKGEPRSS VDDEPIRLPNPMIGFGVPNEPGLITHRSLPELARGPPFFYYENVA LTPKGVWETISRHLFEIPPEFVDSKYFCVAARKRGYIHNLPINNR FQIQPPPKYTIHDAFPLSKRWWPEWDKRTKLNCILTCTGSAQLTN RIRVALEPYNEEPEPPKHVQRYVIDQCKKWNLVWVGKNKAAPLEP DEMESILGFPKNHTRGGGMSRTERFKSLGNSFQVDTVAYHLSVLK PIFPHGINVLSLFTGIGGGEVALHRLQIKMKLVVSVEISKVNRNI LKDFWEQTNQTGELIEFSDIQHLTNDTIEGLMEKYGGEDLVIGGS PCNNLAGGNRVSRVGLEGDQSSLFFEYCRILEVVRARMRGS 619 Arabadopsis CMT1 MAARNKQKKRAEPESDLCFAGKPMSVVESTIRWPHRYQSKKTKLQ APTKKPANKGGKKEDEEIIKQAKCHFDKALVDGVLINLNDDVYVT GLPGKLKFIAKVIELFEADDGVPYCRERWYYRPEDTLIERFSHLV QPKRVFLSNDENDNPLTCIWSKVNIAKVPLPKITSRIEQRVIPPC DYYYDMKYEVPYLNFTSADDGSDASSSLSSDSALNCFENLHKDEK FLLDLYSGCGAMSTGFCMGASISGVKLITKWSVDINKFACDSLKL NHPETEVRNEAAEDELALLKEWKRLCEKFSLVSSTEPVESISELE DEEVEENDDIDEASTGAELEPGEFEVEKFLGIMEGDPQGTGEKTL QLMVRWKGYNSSYDTWEPYSGLGNCKEKLKEYVIDGFKSHLLPLP GTVYTVCGGPPCQGISGYNRYRNNEAPLEDQKNQQLLVELDIIDE LKPNYVLMENVVDLLRESKGFLARHAVASFVAMNYQTRLGMMAAG SYGLPQLRNRVELWAAQPSEKLPPYPLPTHEVAKKENTPKEFKDL QVGRIQMEFLKLDNALTLADAISDLPPVTNYVANDVMDYNDAAPK TEFENFISLKRSETLLPAFGGDPTRRLFDHQPLVLGDDDLERVSY IPKQKGANYRDMPGVLVHNNKAEINPRFRAKLKSGKNVVPAYAIS FIKGKSKKPFGRLWGDEIVNTVVTRAEPHNQCVIHPMQNRVLSVR ENARLQGFPDCYKLCGTIKEKYIQVGNAVAVPVGVALGYAFGMAS QGLTDDEPVIKLPFKYPECMQAKDQI 620 Arabadopsis CMT2 MLSPAKCESEEAQAPLDLHSSSRSEPECLSLVLWCPNPEEAAPSS TRELIKLPDNGEMSLRRSTTLNCNSPEENGGEGRVSQRKSSRGKS QPLLMLTNGCQLRRSPRFRALHANFDNVCSVPVTKGGVSQRKESR GKSQPLLTLTNGCQLRRSPRFRAVDGNFDSVCSVPVTGKFGSRKR KSNSALDKKESSDSEGLTEKDIAVIAKSLEMEIISECQYKNNVAE GRSRLQDPAKRKVDSDTLLYSSINSSKQSLGSNKRMRRSQREMKG TENEGEENLGKSKGKGMSLASCSFRRSTRLSGTVETGNTETLNRR KDCGPALCGAEQVRGTERLVQISKKDHCCEAMKKCEGDGLVSSKQ ELLVFPSGCIKKTVNGCRDRTLGKPRSSGLNTDDIHTSSLKISKN DTSNGLTMTTALVEQDAMESLLQGKTSACGAADKGKTREMHVNST VIYLSDSDEPSSIEYLNGDNLTQVESGSALSSGGNEGIVSLDLNN PTKSTKRKGKRVTRTAVQEQNKRSICFFIGEPLSCEEAQERWRWR YELKERKSKSRGQQSEDDEDKIVANVECHYSQAKVDGHTFSLGDF AYIKGEEEETHVGQIVEFFKTTDGESYFRVQWFYRATDTIMERQA TNHDKRRLFYSTVMNDNPVDCLISKVTVLQVSPRVGLKPNSIKSD YYFDMEYCVEYSTFQTLRNPKTSENKLECCADVVPTESTESILKK KSFSGELPVLDLYSGCGGMSTGLSLGAKISGVDVVTKWAVDQNTA ACKSLKLNHPNTQVRNDAAGDELQLLKEWDKLCKRYVENNDQRTD TLRSVNSTKETSGSSSSSDDDSDSEEYEVEKLVDICFGDHDKTGK NGLKFKVHWKGYRSDEDTWELAEELSNCQDAIREFVTSGFKSKIL PLPGRVGVICGGPPCQGISGYNRHRNVDSPLNDERNQQIIVEMDI VEYLKPSYVLMENVVDILRMDKGSLGRYALSRLVNMRYQARLGIM TAGCYGLSQFRSRVFMWGAVPNKNLPPFPLPTHDVIVRYGLPLEF ERNVVAYAEGQPRKLEKALVLKDAISDLPHVSNDEDREKLPYESL PKTDFQRYIRSTKRDLTGSAIDNCNKRTMLLHDHRPFHINEDDYA RVCQIPKRKGANFRDLPGLIVRNNTVCRDPSMEPVILPSGKPLVP GYVFTFQQGKSKRPFARLWWDETVPTVLTVPTCHSQALLHPEQDR VLTIRESARLQGFPDYFQFCGTIKERYCQIGNAVAVSVSRALGYS LGMAFRGLARDEHLIKLPQNFSHSTYPQLQETIPH 621 Arabadopsis CMT3 MAPKRKRPATKDDTTKSIPKPKKRAPKRAKTVKEEPVTVVEEGEK HVARFLDEPIPESEAKSTWPDRYKPIEVQPPKASSRKKTKDDEKV EIIRARCHYRRAIVDERQIYELNDDAYVQSGEGKDPFICKIIEME EGANGKLYFTARWFYRPSDTVMKEFEILIKKKRVFFSEIQDTNEL GLLEKKLNILMIPLNENTKETIPATENCDFFCDMNYFLPYDTFEA IQQETMMAISESSTISSDTDIREGAAAISEIGECSQETEGHKKAT LLDLYSGCGAMSTGLCMGAQLSGLNLVTKWAVDMNAHACKSLQHN HPETNVRNMTAEDFLELLKEWEKLCIHESLRNSPNSEEYANLHGL NNVEDNEDVSEESENEDDGEVFTVDKIVGISFGVPKKLLKRGLYL KVRWLNYDDSHDTWEPIEGLSNCRGKIEEFVKLGYKSGILPLPGG VDVVCGGPPCQGISGHNRFRNLLDPLEDQKNKQLLVYMNIVEYLK PKFVLMENVVDMLKMAKGYLARFAVGRLLQMNYQVRNGMMAAGAY GLAQFRLRFFLWGALPSEIIPQFPLPTHDLVHRGNIVKEFQGNIV AYDEGHTVKLADKLLLKDVISDLPAVANSEKRDEITYDKDPTTPF QKFIRLRKDEASGSQSKSKSKKHVLYDHHPLNLNINDYERVCQVP KRKGANFRDFPGVIVGPGNVVKLEEGKERVKLESGKTLVPDYALT YVDGKSCKPFGRLWWDEIVPTVVTRAEPHNQVIIHPEQNRVLSIR ENARLQGFPDDYKLFGPPKQKYIQVGNAVAVPVAKALGYALGTAF QGLAVGKDPLLTLPEGFAFMKPTLPSELA 622 Neurospora Rid MAEQNPFVIDDEDDVIQIHDEEEVEEEVAEVIDITEDDIEPSELD RAFGSRPKEETLPSLLLRDQGFIVRPGMTVELKAPIGRFAISFVR VNSIVKVRQAHVNNVTIRGHGFTRAKEMNGMLPKQLNECCLVASI DTRDPRP 623 E. coli strain 12 MNNNDLVAKLWKLCDNLRDGGVSYQNYVNELASLLELKMCKETGQ hsdM EAEYLPEGYRWDDLKSRIGQEQLQFYRKMLVHLGEDDKKLVQAVE HNVSTTITEPKQITALVSNMDSLDWYNGAHGKSRDDEGDMYEGLL QKNANETKSGAGQYFTPRPLIKTIIHLLKPQPREVVQDPAAGTAG FLIEADRYVKSQTNDLDDLDGDTQDFQIHRAFIGLELVPGTRRLA LMNCLLHDIEGNLDHGGAIRLGNTLGSDGENLPKAHIVATNPPFG SAAGTNITRTFVHPTSNKQLCFMQHIIETLHPGGRAAVVVPDNVL FEGGKGTDIRRDLMDKCHLHTILRLPTGIFYAQGVKTNVLEFTKG TVANPNQDKNCTDDVWVYDLRTNMPSFGKRTPFTDEHLQPFERVY GEDPHGLSPRTEGEWSFNAEETEVADSEENKNTDQHLATSRWRKE SREWIRTAKSDSLDISWLKDKDSIDADSLPEPDVLAAEAMGELVQ ALSELDALMRELGASDEADLQRQLLEEAFGGVKE 624 E. coli strain 12 MSAGKLPEGWVIAPVSTVTTLIRGVTYKKEQAINYLKDDYLPLIR hsdS ANNIQNGKFDTTDLVFVPKNLVKESQKISPEDIVIAMSSGSKSVV GKSAHQHLPFECSFGAFCGVLRPEKLIFSGFIAHFTKSSLYRNKI SSLSAGANINNIKPASEDLINIPIPPLAEQKIIAEKLDTLLAQVD STKARFEQIPQILKRFRQAVLGGAVNGKLTEKWRNFEPQHSVEKK LNFESILTELRNGLSSKPNESGVGHPILRISSVRAGHVDQNDIRE LECSESELNRHKLQDGDLLFTRYNGSLEFVGVCGLLKKLQHQNLL YPDKLIRARLTKDALPEYIEIFFSSPSARNAMMNCVKTTSGQKGI SGKDIKSQVVLLPPVKEQAEIVRRVEQLFAYADTIEKQVNNALAR VNNLTQSILAKAFRGELTAQWRAENPDLISGENSAAALLEKIKAE RAASGGKKASRKKS 625 T. aquaticus M TaqI MGLPPLLSLPSNSAPRSLGRVETPPEVVDEMVSLAEAPRGGRVLE PACAHGPFLRAFREAHGTAYRFVGVEIDPKALDLPPWAEGILADE LLWEPGEAFDLILGNPPYGIVGEASKYPIHVFKAVKDLYKKAFST WKGKYNLYGAFLEKAVRLLKPGGVLVFVVPATWLVLEDFALLREF LAREGKTSVYYLGEVFPQKKVSAVVIRFQKSGKGLSLWDTQESES GFTPILWAEYPHWEGEIIRFETEETRKLEISGMPLGDLFHIRFAA RSPEFKKHPAVRKEPGPGLVPVLTGRNLKPGWVDYEKNHSGLWMP KERAKELRDFYATPHLVVAHTKGTRVVAAWDERAYPWREEFHLLP KEGVRLDPSSLVQWLNSEAMQKHVRTLYRDFVPHLTLRMLERLPV RREYGEHTSPESARNE 626 E. coli M EcoDam MKKNRAFLKWAGGKYPLLDDIKRHLPKGECLVEPFVGAGSVELNT DESRYILADINSDLISLYNIVKMRTDEYVQAARELFVPETNCAEV YYQFREEFNKSQDPFRRAVLFLYLNRYGYNGLCRYNLRGEENVPF GRYKKPYFPEAELYHFAEKAQNAFFYCESYADSMARADDASVVYC DPPYAPLSATANFTAYHTNSFTLEQQAHLAEIAEGLVERHIPVLI SNHDTMLTREWYQRAKLHVVKVRRSISSNGGTRKKVDELLALYKP GVVSPAKK 627 C. crescentus M MKFGPETIIHGDCIEQMNALPEKSVDLIFADPPYNLQLGGDLLRP CcrMI DNSKVDAVDDHWDQFESFAAYDKFTREWLKAARRVLKDDGAIWVI GSYHNIFRVGVAVQDLGEWILNDIVWRKSNPMPNEKGTRFANAHE TLIWASKSQNAKRYTENYDALKMANDEVQMRSDWTIPLCTGEERI KGADGQKAHPTQKPEALLYRVILSTTKPGDVILDPFFGVGTTGAA AKRLGRKFIGIEREAEYLEHAKARIAKVVPIAPEDLDVMGSKRAE PRVPFGTIVEAGLLSPGDTLYCSKGTHVAKVRPDGSITVGDLSGS IHKIGALVQSAPACNGWTYWHEKTDAGLAPIDVLRAQVRAGMN 628 C. difficile CamA MDDISQDNFLLSKEYENSLDVDTKKASGIYYTPKIIVDYIVKKTL KNHDIIKNPYPRILDISCGCGNELLEVYDILYDLFEENIYELKKK YDENYWTVDNIHRHILNYCIYGADIDEKAISILKDSLTNKKVVND LDESDIKINLFCCDSLKKKWRYKEDYIVGNPPYIGHKKLEKKYKK FLLEKYSEVYKDKADLYFCFYKKIIDILKQGGIGSVITPRYFLES LSGKDLREYIKSNVNVQEIVDELGANIFKNIGVSSCILTEDKKKT KETYIDVFKIKNEDICINKFETLEELLKSSKFEHFNINQRLLSDE WILVNKDDETFYNKIQEKCKYSLEDIAISFQGIITGCDKAFILSK DDVKLNLVDDKELKCWIKSKNINKYIVDKSEYRLIYSNDIDNENT NKRILDEIIGLYKTKLENRRECKSGIRKWYELQWGREKLFFERKK IMYPYKSNENRFAIDYDNNESSADVYSFFIKEEYLDKESYEYLVG ILNSSVYDKYFKITAKKMSKNIYDYYPNKVMKIRIFRDNNYEEIE NLSKQIISILLNKSIDKGKVEKLQIKMDNLIMDSLGI 629 KAP1 MAASAAAASAAAASAASGSPGPGEGSAGGEKRSTAPSAAASASAS AAASSPAGGGAEALELLEHCGVCRERLRPEREPRLLPCLHSACSA CLGPAAPAAANSSGDGGAAGDGTVVDCPVCKQQCFSKDIVENYFM RDSGSKAATDAQDANQCCTSCEDNAPATSYCVECSEPLCETCVEA HQRVKYTKDHTVRSTGPAKSRDGERTVYCNVHKHEPLVLFCESCD TLTCRDCQLNAHKDHQYQFLEDAVRNQRKLLASLVKRLGDKHATL QKSTKEVRSSIRQVSDVQKRVQVDVKMAILQIMKELNKRGRVLVN DAQKVTEGQQERLERQHWTMTKIQKHQEHILRFASWALESDNNTA LLLSKKLIYFQLHRALKMIVDPVEPHGEMKFQWDLNAWTKSAEAF GKIVAERPGTNSTGPAPMAPPRAPGPLSKQGSGSSQPMEVQEGYG FGSGDDPYSSAEPHVSGVKRSRSGEGEVSGLMRKVPRVSLERLDL DLTADSQPPVEKVFPGSTTEDYNLIVIERGAAAAATGQPGTAPAG TPGAPPLAGMAIVKEEETEAAIGAPPTATEGPETKPVLMALAEGP GAEGPRLASPSGSTSSGLEVVAPEGTSAPGGGPGTLDDSATICRV CQKPGDLVMCNQCEFCFHLDCHLPALQDVPGEEWSCSLCHVLPDL KEEDGSLSLDGADSTGVVAKLSPANQRKCERVLLALFCHEPCRPL HQLATDSTFSLDQPGGTLDLTLIRARLQEKLSPPYSSPQEFAQDV GRMFKQFNKLTEDKADVQSIIGLQRFFETRMNEAFGDTKESAVLV EPPPMSLPGAGLSSQELSGGPGDGP 630 MECP2 MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEP VQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRD RGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSK VELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPK APGTGRGRGRPKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMP FQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAIPKKRGR KPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIE VKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASS PPKKEHHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEP QDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKY KHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS 631 linker SGGS 532 linker SGGSSGSETPGTSESATPESSGGS 633 linker SGGSSGGSSGSETPGTSESATPESSGGSSGGS 634 linker GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGSEPATSGGSGGS 635 G linker GSGGG 636 GX4 linker GGGGSGGGGSGGGGSGGGGS 637 W linker SSGNSNANSRGPSFSSGLVPLSLRGSH 638 XTEN linker SGSETPGTSESATPES (XTEN16) 639 XTEN linker SGGSSGGSSGSETPGTSESATPES 640 XTEN linker SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS 641 XTEN linker SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSET PGTSESATPESSGGSSGGS 642 XTEN linker PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPA TS 643 XTEN linker GGPSSGAPPPSGGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG (XTEN80) SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE 644 NLS PKKKRKV 645 NLS AVKRPAATKKAGQAKKKKLD 646 NLS MSRRRKANPTKLSENAKKLAKEVEN 647 NLS PAAKRVKLD 648 NLS KLKIKRPVK 649 NLS MDSLLMNRRKFLYQFKNVRWAKGRRETYLC 660 fusion protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVL (Configuration 7) SLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIM YVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGT GRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRE LESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQE CLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEKEDILWCT EMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKE YFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEP SMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGS LQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGE TLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPS SRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVL SLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPF DLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWM FVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIP AIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCELPLRE YFKYFSTELTSSLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH SIKKNLIGALLEDSGETAEATRLKRTARRRYTRRKNRICYLQEIF SNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEK YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNP DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRR LENLIAQLPGEKKNGLFGNLIALSLGLTPNEKSNEDLAEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTF RIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSE IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLEKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRENASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLE EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDELKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSE SATPESRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKN LVSLGYQLTKPDVILRLEKGEEPSADYKDDDDKAPKKKRKVPKKK RKV 661 fusion protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVL (Configuration 9) SLEDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIM YVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGT GRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRE LESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQE CLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEKEDILWCT EMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKE YFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEP SMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGS LQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGE TLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPS SRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVL SLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPF DLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWM FVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIP AIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLRE YFKYFSTELTSSLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEK YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNP DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRR LENLIAQLPGEKKNGLFGNLIALSLGLTPNEKSNEDLAEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTE RIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSE IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRENASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LEVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYEDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSE SATPESTGNKKLEAVGTGIEPKAMSQGLVTFGDVAVDESQEEWEW LNPIQRNLYRKVMLENYRNLASLGLCVSKPDVISSLEQGKEPWSA DYKDDDDKAPKKKRKVPKKKRKV 662 fusion protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVL (Configuration 11) SLEDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIM YVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGT GRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRE LESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQE CLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEKEDILWCT EMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKE YFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEP SMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGS LQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGE TLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPS SRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVL SLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPF DLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWM FVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIP AIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCFLPLRE YFKYFSTELTSSLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIF SNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEK YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNP DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRR LENLIAQLPGEKKNGLFGNLIALSLGLTPNEKSNEDLAEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPELKDNREKIEKILTF RIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSE IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLEKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDFLKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LEVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYEDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSE SATPESTGDSVAFEDVAVNETLEEWALLDPSQKNLYRDVMRETER NLASVGKQWEDQNIEDPFKIPRRNISHIPERLCESKEGGQGEESA DYKDDDDKAPKKKRKVPKKKRKV 663 fusion protein MGTMPKKKRKVPKKKRKVYNHDQEFDPPKVYPPVPAEKRKPIRVL (Configuration 13) SLEDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIM YVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGT GRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVSDKRDISRF LESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQE CLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVEMNEKEDILWCT EMERVFGFPVHYTDVSNMSRLARQRLLGRSWSVPVIRHLFAPLKE YFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMAAIPALDPEAEP SMDVILVGSSELSSSVSPGTGRDLIAYEVKANQRNIEDICICCGS LQVHTQHPLFEGGICAPCKDKFLDALFLYDDDGYQSYCSICCSGE TLLICGNPDCTRCYCFECVDSLVGPGTSGKVHAMSNWVCYLCLPS SRSGLLQRRRKWRSQLKAFYDRESENPLEMFETVPVWRRQPVRVL SLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPF DLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWM FVDNLVLNKEDLDVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIP AIRSRHWALVSEEELSLLAQNKQSSKLAAKWPTKLVKNCELPLRE YFKYFSTELTSSLGGPSSGAPPPSGGSPAGSPTSTEEGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSELEDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRH SIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIF SNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNIVDEVAYHEK YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKERGHFLIEGDLNP DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRR LENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNEDLAEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTF RIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSE IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIS GVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLEDDKVMKQLKRRRYTGWGRLSRKLINGIR DKQSGKTILDELKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQ GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT KYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK ATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRK RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT LIHQSITGLYETRIDLSQLGGDSPKKKRKVGVDGSSGSETPGTSE SATPESTGMNNSQGRVTFEDVTVNFTQGEWQRLNPEQRNLYRDVM LENYSNLVSVGQGETTKPDVILRLEQGKEPWLEEEEVLGSGRAEK NGDIGGQIWKPKDVKESLSADYKDDDDKAPKKKRKVPKKKRKV 664 linker GGGGS 665 linker EAAAK 631 linker SGGS

Claims

1. A system for repressing transcription of a human PCSK9 gene in a human cell, optionally a human hepatocyte, comprising

a) one or more fusion proteins that collectively comprise a DNA methyltransferase (DNMT) domain and/or a domain that recruits a DNMT, optionally wherein the DNMT domain and/or the recruiter domain comprise a DNMT3A domain and/or a DNMT3L domain, and optionally wherein the recruited DNMT is DNMT3A, and a transcriptional repressor domain, each domain being linked to a DNA-binding domain that binds to a target region in the human PCSK9 gene; or

b) one or more nucleic acid molecules encoding the one or more fusion proteins.

2. The system of claim 1, wherein the DNA-binding domain binds to a target sequence in SEQ ID NO: 1488 or 1489.

3. The system of claim 1 or 2, wherein the DNA-binding domain targets the fusion protein(s) to one or more sequences in the PCSK9 gene selected from SEQ ID NOs: 700-747 and 1036-1261.

4. The system of any one of claims 1-3, wherein the DNA-binding domain comprises a dead CRISPR Cas (dCas) domain, a ZFP domain, or a TALE domain.

5. The system of claim 4, wherein the DNA-binding domain comprises a dCas9 domain and the system further comprises (i) one or more guide RNAs comprising any one of SEQ ID NOs: 1262-1487, or (ii) nucleic acid molecules coding for the one or more guide RNAs.

6. The system of claim 4 or 5, wherein the dCas domain comprises a dCas9 sequence, optionally a sequence with at least 90% identity to SEQ ID NO: 12 or 13.

7. The system of claim 4, wherein the ZFP domain targets a nucleotide sequence selected from SEQ ID NOs: 700-747.

8. The system of claim 7, wherein the ZFP domain comprises the F1-F6 amino acid sequences of any one of ZF001 through ZF048 as shown in Table 1.

9. The system of any one of claims 1-8, wherein the DNMT3A domain comprises a sequence with at least 90% identity to SEQ ID NO: 574 or 575.

10. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 578-581.

11. The system of any one of claims 1-9, wherein the DNMT3L domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 582-603.

12. The system of any one of claims 1-8, wherein the DNMT domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 601-603.

13. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 33-570.

14. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a KRAB domain derived from KOX1, ZIM3, ZFP28, or ZN627.

15. The system of claim 14, wherein the KRAB domain comprises a sequence with at least 90% identity to a sequence selected from SEQ ID NOs: 89, 116, 245, and 255.

16. The system of any one of claims 1-12, wherein the transcriptional repressor domain comprises a fusion of the N- and C-terminal regions of ZIM3 and KOX1 KRAB, and optionally comprises the amino acid sequence of SEQ ID NO: 571 or 572.

17. The system of any one of claims 1-12, wherein the transcriptional repressor domain is derived from KAP1, MECP2, HP1a/CBX5, HP1b, CBX8, CDYL2, TOX, TOX3, TOX4, EED, EZH2, RBBP4, RCOR1, or SCML2.

18. The system of any one of claims 1-17, wherein the system comprises

a) a fusion protein comprising the DNMT3A domain, the DNMT3L domain, the transcriptional repressor domain, and the DNA-binding domain, optionally wherein one or both of the DNMT3A domain and the DNMT3L domain are human, and optionally wherein the DNA-binding domain is a dead CRISPR Cas domain or a ZFP domain; or

b) a nucleic acid molecule encoding the fusion protein.

19. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, the DNMT3A domain, a first peptide linker, the DNMT3L domain, a second peptide linker, the DNA-binding domain, a third peptide linker, and the transcriptional repressor domain.

20. The system of claim 19, wherein the fusion protein comprises, from N-terminus to C-terminus, the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, a first nuclear localization signal (NLS), the DNA-binding domain, a second NLS, the third peptide linker, and the transcriptional repressor domain.

21. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a first nuclear localization signal (NLS), the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and a second NLS.

22. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second nuclear localization signals (NLSs), the DNMT3A domain, the first peptide linker, the DNMT3L domain, the second peptide linker, the DNA-binding domain, the third peptide linker, the transcriptional repressor domain, and third and fourth NLSs.

23. The system of any one of claims 18-22, wherein the transcriptional repressor domain is a KRAB domain, optionally a human KOX1, ZFP28, ZN627, or ZIM3 KRAB domain.

24. The system of any one of claims 19-23, wherein one or both of the second and third peptide linkers are XTEN linkers, optionally selected from XTEN80 and XTEN16, and further optionally wherein the second peptide linker is XTEN80, and the third peptide linker is XTEN16.

25. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a dSpCas9 domain, a second NLS, an XTEN16 peptide linker, and a human KOX1 KRAB domain.

26. The system of claim 25, wherein the fusion protein comprises SEQ ID NO: 658 or a sequence at least 90% identical thereto, or SEQ ID NO: 1495 or a sequence at least 90% identical thereto.

27. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a first NLS, a ZFP domain, a second NLS, an XTEN16 linker, and a human KOX1 KRAB domain.

28. The system of claim 27, wherein the fusion protein comprises SEQ ID NO: 659 or a sequence at least 90% identical thereto, or SEQ ID NO: 1496 or a sequence at least 90% identical thereto, optionally wherein the ZFP comprises the F1-F6 amino acid sequences of any one of ZF001 through ZF048 as shown in Table 1.

29. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs.

30. The system of claim 29, wherein the fusion protein comprises SEQ ID NO: 660 or a sequence at least 90% identical thereto.

31. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human KOX1 KRAB domain, and third and fourth NLSs, optionally wherein the fusion protein comprises SEQ ID NO: 1514 or a sequence at least 90% identical thereto, or SEQ ID NO: 1523 or a sequence at least 90% identical thereto.

32. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.

33. The system of claim 32, wherein the fusion protein comprises SEQ ID NO: 661 or a sequence at least 90% identical thereto, or SEQ ID NO: 1516 or a sequence at least 90% identical thereto.

34. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZFP28 KRAB domain, and third and fourth NLSs.

35. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.

36. The system of claim 35, wherein the fusion protein comprises SEQ ID NO: 662 or a sequence at least 90% identical thereto, or SEQ ID NO: 1520 or a sequence at least 90% identical thereto.

37. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZN627 KRAB domain, and third and fourth NLSs.

38. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a dSpCas9 domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.

39. The system of claim 38, wherein the fusion protein comprises SEQ ID NO: 663 or a sequence at least 90% identical thereto or SEQ ID NO: 1518 or a sequence at least 90% identical thereto.

40. The system of claim 18, wherein the fusion protein comprises, from N-terminus to C-terminus, first and second NLSs, a human DNMT3A domain, a first peptide linker, a human DNMT3L domain, an XTEN80 peptide linker, a ZFP domain, an XTEN16 peptide linker, a human ZIM3 KRAB domain, and third and fourth NLSs.

41. The system of any one of claims 20-40, wherein at least one of the NLSs is an SV40 NLS.

42. The system of any one of claims 1-17, wherein the system comprises:

a) a first fusion protein comprising a first DNA-binding domain and comprising or recruiting the DNMT3A domain, a second fusion protein comprising a second DNA-binding domain and comprising or recruiting the DNMT3L domain, and a third fusion protein comprising a third DNA-binding domain and comprising or recruiting the transcriptional repressor domain; or

b) one or more nucleic acid molecules encoding the fusion proteins.

43. A human cell comprising the system of any one of claims 1-42, or progeny of the cell, optionally wherein the cell is a hepatocyte.

44. A pharmaceutical composition comprising the system of any one of claims 1-42 and a pharmaceutically acceptable excipient, optionally wherein

the composition comprises lipid nanoparticles (LNPs) comprising the system, and/or

the DNA-binding domain is a dCas domain and the LNPs further comprise one or more gRNAs.

45. A method of treating a patient in need thereof, comprising administering the system of any one of claims 1-42 or the pharmaceutical composition of claim 44 to the patient, optionally intravenously.

46. The method of claim 45, wherein the patient

has heart disease,

has elevated low-density lipoprotein cholesterol (LDL-C) or hypercholesterolemia,

is at risk of developing myocardial infarction, stroke, or unstable angina, and/or

has primary hyperlipidemia, optionally heterozygous familial hypercholesterolemia (HeFH), or homozygous familial hypercholesterolemia (HoFH).

47. The system of any one of claims 1-42, or the pharmaceutical composition of claim 44, for use in treating a patient in need thereof, optionally in the method of claim 45 or 46.

48. Use of the system of any one of claims 1-42 in the manufacture of a medicament for treating a patient in need thereof, optionally in the method of claim 45 or 46.